Blackcurrant (Ribes nigrum L.) Seeds-A Valuable Byproduct for Further Processing.

The rational exploitation of byproducts is important from the point of view of their potential applicability in various fields. In this study, the possibility of further processing of blackcurrant seeds (BCs), which are a byproduct of fruit processing, was investigated. BCs were used as a material for the extraction of oil on a semi-industrial scale, and the residues were assessed in terms of their potential application in skin care products. Supercritical fluid extraction (SFE) using CO2 at pressures of 230 and 330 bar and extraction temperature of 40 °C was exploited for isolation of oil, and the products were characterised taking into account lipophilic constituents. After 120 min, the oil yields were 19.67% and 20.94% using CO2 at 230 and 330 bar, respectively, which showed that SFE was an effective method on a semi-industrial scale, taking into account the extraction yield. The oils had similar fatty acid compositions with a high percentage of linoleic acid (ca. 43%); however, tocopherols and carotenoids were most abundant in the oil obtained at 230 bar. It was also found that the composition of the SFE oils was comparable with that of cold-pressed oil, which shows that supercritical fluid extraction provides a high-quality product; therefore, it can be an alternative to cold pressing. Furthermore, the chemical compositions of the extracts from the oil isolation residues were established using UPLC-MS, and the impact of the extracts on human skin fibroblasts was assessed using the MTT and NR assays. The quantitative analysis revealed that the residues contained high amounts of polyphenolic acids, including gallic, protocatechuic, and hydroxybenzoic acid derivatives, as well as flavonoids, especially quercetin and kaempferol glucoside. Moreover, it was found that the extracts were nontoxic and exerted a stimulatory effect on cell metabolism. Therefore, they can be a valuable additive to natural plant-based cosmetics. Our results showed that blackcurrant seeds, regarded as a byproduct, can be a valuable material for further use.

Shotgun Lipidomic Analysis for Differentiation of Niche Cold Pressed Oils.

The fast-growing food industry is bringing significant number of new products to the market. To protect consumers' health and rights, it is crucial that food control laboratories are able to ensure reliable quality testing, including product authentication and detection of adulterations. In our study, we applied a fast and eco-friendly method based on shotgun-lipidomic mass spectrometry for the authentication of niche edible oils. Comprehensive lipid profiles of camelina (CA), flax (FL) and hemp (HP) seed oils were obtained. With the aid of principal component analysis (PCA), it was possible to detect and distinguish each of them based on their lipid profiles. Lipidomic markers characteristic ofthe oils were also identified, which can be used as targets and expedite development of new multiplexed testing methods.

Paraoxonase 1 Phenotype and Protein N-Homocysteinylation in Patients with Rheumatoid Arthritis: Implications for Cardiovascular Disease.

Paraoxonase 1 (PON1) is the high density lipoprotein-associated esterase which inhibits the development of atherosclerosis by metabolizing lipid peroxidation products as well as hydrolyzing proatherogenic metabolite of homocysteine (Hcy), Hcy thiolactone, which otherwise reacts with lysine groups of proteins, thus forming N-Hcy-protein in a process referred to as protein N-homocysteinylation. Rheumatoid arthritis (RA) is the chronic inflammatory autoimmune disease associated with increased risk of cardiovascular complications, but the underlying mechanisms are incompletely understood. We examined PON1 status and N-homocysteinylation of serum proteins in patients with RA. Blood was collected from 74 RA patients and 70 control subjects. PON1 activity was measured toward synthetic (paraoxon, phenyl acetate) and natural (Hcy thiolactone) substrates. PON1 protein concentration was measured by ELISA. Total Hcy as well as N-Hcy-protein were measured in serum as well. PON1 activity toward Hcy thiolactone was lower in RA patients than in control subjects which was accompanied by increased concentration of N-Hcy-protein despite normal total Hcy concentration. PON1 protein concentration was unchanged in the RA group, but the specific enzyme activity was reduced. When RA patients were categorized according to the DAS28-ESR score, PON1 concentration and enzymatic activity were lower whereas N-Hcy-protein was higher in those with high disease activity. PON1 activity and Hcy thiolactone were correlated with DAS28-ESR score and myeloperoxidase concentration. In conclusion, RA is associated with deficiency of PON1 activity and increased protein N-homocyseinylation which may contribute to accelerated development of cardiovascular diseases.

Cladribine Treatment Improved Homocysteine Metabolism and Increased Total Serum Antioxidant Activity in Secondary Progressive Multiple Sclerosis Patients.

Hyperhomocysteinemia plays a crucial role in the pathogenesis of many diseases of the central nervous system (CNS). The nervous system is particularly sensitive to high homocysteine (Hcy) level mainly due to its prooxidative and cytotoxic effects. Cladribine, a drug recently registered for the treatment of multiple sclerosis (MS), possesses additionally neuroprotective effects which are independent of its peripheral immunosuppressant action. Accumulating evidence suggests that oxidative stress and homocysteine thiolactone-mediated protein homocysteinylation play a causal role in MS. Both of these processes may be attenuated by paraoxonase 1 (PON1). Therefore, in the present study, we aimed to examine whether the beneficial effects of the drug in MS patients with a secondary progressive (SP) clinical course, treated with cladribine subcutaneously (s.c.), may be related to its ability to modify serum PON1 activity, Hcy concentration, and protein homocysteinylation, as well as to correct total antioxidant status. A total of 118 subjects were enrolled into the study: (1) patients with a SP type of MS, SP-MS (n = 40); (2) patients with a relapsing-remitting (RR) type of MS, RR-MS (n = 30); and (3) healthy people (n = 48). Patients with SP-MS were treated with cladribine. The drug was given in SP-SM patients s.c. six times every 6 weeks up to a total mean cumulative dose of 1.8 mg/kg. PON1 activity was assessed spectrophotometrically. The level of Hcy, homocysteine thiolactone (HTL) attached to plasma proteins (N-Hcy-protein), and antibodies against homocysteinylated proteins was assessed with an enzyme immunoassay. The total antioxidant activity of the serum was assessed with the ferric-reducing activity of plasma (FRAP) method. Basically, there was no difference in PON1 activity between untreated SP-MS, RR-MS, and control subjects. Serum Hcy was significantly higher in RR-MS patients (p < 0.001) and in SP-MS patients (p < 0.01) compared to the control group. The N-Hcy protein level was higher in RR-MS patients (p < 0.05) in comparison to the control group. Moreover, the elevated level of antibodies against homocysteinylated proteins was observed in the serum of patients with SP-MS. The total antioxidant capacity of serum was lower in MS patients vs. the control group (p < 0.001). After cladribine treatment, the activity of PON1 did not change in SP-MS patients, whereas cladribine treatment decreased the level of total Hcy (p < 0.05). Treatment with cladribine increased the total serum antioxidant activity in SP-MS patients (p < 0.01). The Expanded Disability Status Scale (EDSS) score did not change in SP-MS patients. Cladribine treatment in the SP-MS group attenuates hyperhomocysteinemia-induced protein homocysteinylation (n.s.). It also stabilises the neurological condition of SP-MS patients. The stabilisation of a neurological condition observed in SP-MS patients after cladribine treatment may be partially related to its ability to reduce elevated Hcy level and to improve serum antioxidant potential.

The effect of exenatide (a GLP-1 analog) and sitagliptin (a DPP-4 inhibitor) on plasma platelet-activating factor acetylhydrolase (PAF-AH) activity and concentration in normal and fructose-fed rats.

Inflammation and oxidative stress are the two processes crucial in atherogenesis. Platelet-activating factor acetylhydrolase (PAF-AH), a plasma lipoprotein-associated enzyme, degrades pro-inflammatory lipids generated within oxidatively modified lipoproteins. Extensive evidence shows that incretin-based drugs, a new class of anti-diabetic agents, can provide cardiovascular protection that cannot be attributed to their glucose-lowering effects. The present study was undertaken to determine whether the antiatherogenic effects of the GLP-1(glucagon-like peptide-1) receptor agonist (exenatide) and DPP-4(dipeptidyl peptidase-4) inhibitors (sitagliptin) may occur via the regulation of platelet-activating factor acetylhydrolase (PAF-AH) activity/mass and inhibition of low-density lipoprotein (LDL) oxidation in the fructose-fed rats. Normal and fructose-fed rats (8 wk) were treated (4 wk) with sitagliptin (5 and 10 mg/kg p.o.) or with exenatide (5 and 10 µg/kg, s.c.). Plasma PAF-AH activity and phosphatidylcholine (PC) concentration were measured colorimetrically. Plasma PAF-AH concentration, oxidized LDL (oxLDL), hexanoyl-Lys adduct (HEL), lyso-PC, apolipoprotein A-I (apoA-I), apoB, platelet-activating factor (PAF), monocyte chemoattractant protein-1 (MCP-1) and endothelin-1 (ET-1) were measured by ELISA. The four-week exenatide (5 µg/kg, sc.) treatment of fructose fed-rats significantly increased plasma PAF-AH activity (+33%, P < 0.001) and decreased the level of circulating oxLDL (-42%, P < 0.05) and MCP-1 (-23%, P < 0.01). These changes were accompanied by the decrease in plasma PC/lyso-PC (-47%, P < 0.001) and apoB/apoA-I ratio (-75%, P < 0.001). The effect of exenatide on enzyme activity was associated with only a minor effect on metabolic parameters and was independent of weight reduction. Exenatide but not sitagliptin inhibits oxidative modification of LDL probably due to favorable effect on plasma PAF-AH activity.

Metabolic Effects of Metformin in the Failing Heart.

Accumulating evidence shows that metformin is an insulin-sensitizing antidiabetic drug widely used in the treatment of type 2 diabetes mellitus (T2DM), which can exert favorable effects on cardiovascular risk and may be safely used in patients with heart failure (HF), and even able to reduce the incidence of HF and to reduce HF mortality. In failing hearts, metformin improves myocardial energy metabolic status through the activation of AMP (adenosine monophosphate)-activated protein kinase (AMPK) and the regulation of lipid and glucose metabolism. By increasing nitric oxide (NO) bioavailability, limiting interstitial fibrosis, reducing the deposition of advanced glycation end-products (AGEs), and inhibiting myocardial cell apoptosis metformin reduces cardiac remodeling and hypertrophy, and thereby preserves left ventricular systolic and diastolic functions. While a lot of preclinical and clinical studies showed the cardiovascular safety of metformin therapy in diabetic patients and HF, to confirm observed benefits, the specific large-scale trials configured for HF development in diabetic patients as a primary endpoints are necessary.

Hydrogen sulfide in the regulation of insulin secretion and insulin sensitivity: Implications for the pathogenesis and treatment of diabetes mellitus.

Insulin secretion and sensitivity play an essential role in maintaining normal glucose level and their abnormalities result in diabetes mellitus. H2S-synthesizing enzymes, CBS and/or CSE, are expressed in insulin-secreting pancreatic ß cells and H2S inhibits insulin secretion by activating ATP-sensitive K+ channels. In addition, H2S has been reported to have either pro- or antiapoptotic effects on ß cells. Studies in the animal models suggest that excess of H2S in pancreatic islets may contribute to both type 1 and type 2 diabetes. H2S has also been demonstrated to regulate insulin sensitivity. In the liver, H2S stimulates gluconeogenesis and glycogenolysis and inhibits glucose utilization and glycogen storage. Its effect on insulin-stimulated glucose uptake in the adipose tissue is controversial; both stimulation and inhibition have been reported. H2S may also regulate adipose tissue lipolysis, adipokine production and inflammation; the processes important for local and systemic insulin sensitivity. Little is known about the effect of H2S on skeletal muscle metabolism. High fat diet, obesity and insulin resistance affect CBS/CSE/H2S system in the liver and adipose tissue, although the effect depends on diet composition, animals species and time of high-fat feeding. Most studies indicate that blood H2S concentration decreases in animal models of diabetes and in diabetic humans.

The pathophysiological basis of the protective effects of metformin in heart failure.

Metformin, currently recommended as the drug of first choice in type 2 diabetes mellitus (T2DM), is one of the few antihiperglycemic drugs to reduce cardiovascular risk. Nonetheless, due to the risk of lactic acidosis during metformin therapy, its usage in patients with diabetes and heart failure (HF) is still a matter of debate. The aim of this review is to present data supporting the possibility of using metformin in the treatment of diabetic patients with concomitant heart failure. In the failing heart, metformin through the mechanism related to AMP-activated protein kinase (AMPK) activity, improves free fatty acids (FFA) and glucose metabolism, mitochondrial biogenesis, as well as nitric oxide (NO)-NO synthase pathway. Metformin can also inhibit the generation and accumulation of advanced glycation end products (AGEs) and thereby prevents the development of the adverse structural and functional changes in myocardium.In summary, experimental and clinical data indicate the ability of metformin to prevent the development of the structural and functional changes in myocardium, although further basic research and clinical studies assessing benefits and safety of metformin therapy in patients with HF are required.

The paraoxonase 1 (PON1), platelet-activating factor acetylohydrolase (PAF-AH) and dimethylarginine dimethylaminohydrolase (DDAH) activity in the metformin treated normal and diabetic rats.

Antidiabetic agents per se, apart from their glucose-lowering effect, can have an important impact on modifying the cardiovascular risk. The present study was undertaken to determine whether the known cardio-protective effects of metformin are linked to its potential ability to affect activities of HDL's paraoxonase (PON1) and platelet activating factor acetylohydrolase (PAF-AH) or via its interaction with the asymmetric dimethylarginine (ADMA)- dimethylarginine dimethylaminohydrolase (DDAH) axis. Normal and streptozotocin (STZ)-induced diabetic rats were treated with metformin (300mg/kg; 4 weeks). The activity of PON1, PAF-AH and DDAH were measured spectrophotometrically. The plasma ADMA level was determined by ELISA method. In STZ-induced diabetic rats the long-term administration of metformin normalized reduced PON1 activity assayed toward paraoxon (+42.5%, P<0.05), phenyl acetate (+22.35%, P<0.05) and γ-decanolactone (+108.0%, P<0.01), without affecting elevated PAF-AH activity in the plasma. Moreover, metformin increased DDAH activity in the renal cortex (+38.24%, P<0.01). Additionally metformin administration caused the increase in PON1 activity in the liver (+29.2%, P<0.01) accompanied by the reduction in the lipid peroxidation (-59.8%, P<0.001). Similarly, in non-diabetic treated rats the increase in liver PON1 activity was observed toward both paraoxon (+80.19%, P<0.001) and phenyl acetate (+29.3%, P<0.05), respectively. The present study has demonstrated that insulin-sensitizer metformin is important for preserving antioxidant HDL function in diabetes. Metformin might also exert its effect against diabetic complications by improving DDAH activity in the kidney and increasing PON1 activity in the liver.

Differential effects of statins on endogenous H2S formation in perivascular adipose tissue.

Hydrogen sulfide (H(2)S) is a new gasotransmitter synthesized enzymatically from l-cysteine in cytosol and is oxidized in mitochondria. In the cardiovascular system, H(2)S regulates vascular tone, inhibits atherogenesis, and protects against myocardial ischemia-reperfusion injury. We examined the effect of statins on vascular H(2)S production. Male Wistar rats received pravastatin (40mg/kg/day) or atorvastatin (20mg/kg/day) for 3 weeks and then H(2)S formation was measured in aortic media, periaortic adipose tissue (PAAT) and the liver. Only atorvastatin increased H(2)S production in PAAT whereas both statins stimulated its formation in the liver. Neither statin affected H(2)S production in aortic media. H(2)S formation in post-mitochondrial supernatant was higher than in mitochondria-containing supernatant and was not influenced by statins in any tissue. In addition, oxidation of exogenous H(2)S in isolated liver mitochondria was slower in statin-treated than in control rats. These data indicate that statins increase net H(2)S production by inhibiting its mitochondrial oxidation. Statins had no effect on the activity of H(2)S-metabolizing enzyme, sulfide:quinone oxidoreductase, measured at saturating coenzyme Q concentration. Both statins reduced CoQ(9) concentration in plasma and liver, but only atorvastatin decreased CoQ(9) in PAAT. Atorvastatin attenuated phenylephrine-induced contraction of PAAT+ but not of PAAT- aortic rings. Effects of atorvastatin on net H(2)S production, mitochondrial H(2)S oxidation and aortic contractility were abolished by supplementation of exogenous CoQ(9). In conclusion, lipophilic atorvastatin, but not hydrophilic pravastatin, increases net H(2)S production in perivascular adipose tissue by inhibiting its mitochondrial oxidation. This effect is mediated by statin-induced CoQ(9) deficiency and results in the augmentation of anticontractile effect of perivascular adipose tissue.

Relation between markers of inflammation and estradiol in older men.

BACKGROUND: Inflammation plays a key role in the development of atherosclerosis. Studies in women receiving estrogens show their proinflammatory effects. This study sought to determine relation between sex hormones and 2 inflammation markers: C-reactive protein and fibrinogen. MATERIAL/METHODS: One hundred men of at least age 50 years were enrolled in the study. Plasma levels of total testosterone, estradiol, sex hormone binding globulin, high-sensitivity C-reactive protein, and fibrinogen were measured. Free estradiol and free testosterone were calculated. RESULTS: Estradiol and free estradiol levels were positively correlated with C-reactive protein and fibrinogen. In a subgroup analysis, this association persisted only in patients with stable coronary artery disease. No significant correlations were found between testosterone, free testosterone, sex hormone binding globulin, and markers of inflammation. CONCLUSIONS: This study suggests that estradiol may have proinflammatory effects in older men with coronary artery disease.

Prospective evaluation of the outcome of velopharyngeal insufficiency therapy after simultaneous double z-plasty and sphincter pharyngoplasty.

OBJECTIVE: One of the main goals in the management of cleft palate is to achieve a good quality of speech. The aim of the prospective study was to evaluate the effectiveness of therapy in patients with velopharyngeal insufficiency treated by simultaneously performed Furlow and Orticochea operations. PATIENTS AND METHODS: From May 2007 to May 2008 we treated 14 consecutive patients (6 males and 8 females, mean age 14 years). The indications for surgery were based on nasofiberscopic examination, evaluation of speech quality, nasometry and morphology of the palate. The velopharyngeal closure was below 80% in all the patients; they had pronounced nasality and limited intelligibility of speech. All the palates were short. RESULTS: The final outcome of treatment was based on the combined evaluation of 4 parameters: closure, speech intelligibility, nasality and the nasalance index. Ten patients achieved full recovery (71%), the remaining 4 had improved recovery (29%). CONCLUSION: A 1-stage Furlow operation and sphincter pharyngoplasty are an effective modality in the therapy of velopharyngeal insufficiency. Indications for posterior pharyngeal flap pharyngoplasty should be limited to the cases in which a simultaneous Furlow operation and sphincter pharyngoplasty are not possible due to a deficit of the palatine tissue.

The differentiating effect of glimepiride and glibenclamide on paraoxonase 1 and platelet-activating factor acetylohydrolase activity.

AIMS: The present study was designed to examine the effect of sulphonylureas, glimepiride (GM) and glibenclamide (GB), on paraoxonase 1 (PON1) and platelet activating factor acetylohydrolase (PAF-AH) activity in normal and streptozotocin (STZ)-induced (50 mg/kg) diabetic rats. MAIN METHODS: In treated groups, glimepiride (0.1 mg/kg) or glibenclamide (2 mg/kg) was given orally for 4 weeks. A PON1 and PAF-AH activity were estimated by spectrophotometric method. KEY FINDINGS: Hyperglycemia was accompanied by a significant decrease in plasma PON1 activity toward paraoxon (P < 0.001) and phenyl acetate (P < 0.01) and increase in plasma PAF-AH activity (P < 0.01). In STZ-induced diabetic rats the administration of both GM and GB had no effect on plasma PON1 activity but reversed elevated plasma PAF-AH activity (GM: P < 0.05, GB: P < 0.01). In non-diabetic rats after either GM or GB administration the decreased PON1 activity in the plasma was observed (GM: P < 0.001, GB: P < 0.05), but plasma PAF-AH activity remained unchanged. Both GM and GB had no effect on total plasma antioxidant capacity in diabetic and control treated groups. Additionally, both drugs increased PON1 activity toward phenyl acetate in the liver, in diabetic rats (GM: P < 0.05, GB:ns) as well as in non-diabetic rats (GM: P < 0.001, GB: P < 0.001), and reduced lipid peroxidation in the liver. SIGNIFICANCE: These results demonstrate that in streptozotocin-induced diabetic rats as well as in normal rats glimepiride and glibenclamide have no beneficial effects on circulating PON1 and PAF-AH activities, but both drugs increase PON1 activity in the liver.

Chronic hyperleptinemia induces resistance to acute natriuretic and NO-mimetic effects of leptin.

Apart from controlling energy balance, leptin, secreted by adipose tissue, is also involved in the regulation of cardiovascular function. Previous studies have demonstrated that acutely administered leptin stimulates natriuresis and vascular nitric oxide (NO) production and that these effects are impaired in obese animals. However, the mechanism of resistance to leptin is not clear. Because obesity is associated with chronically elevated leptin, we examined if long-term hyperleptinemia impairs acute effects of leptin on sodium excretion and NO production in the absence of obesity. Hyperleptinemia was induced in lean rats by administration of exogenous leptin at a dose of 0.5mg/kg/day for 7 days, and then acute effect of leptin (1mg/kg i.v.) was studied under general anesthesia. Leptin increased fractional sodium excretion and decreased Na(+),K(+)-ATPase activity in the renal medulla. In addition, leptin increased the level of NO metabolites and cyclic GMP in plasma and aortic wall. These acute effects of leptin were impaired in hyperleptinemic animals. In both control and hyperleptinemic groups the effect of leptin on Na(+) excretion and renal Na(+),K(+)-ATPase was abolished by phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, but not by protein kinase B/Akt inhibitor, triciribine,. In contrast, acute effect of leptin on NO metabolites and cGMP was abolished by triciribine but not by wortmannin. Leptin stimulated Akt phosphorylation at Ser(473) in aortic tissue but not in the kidney, and this effect was comparable in control and hyperleptinemic groups. These results suggest that hyperleptinemia may mediate "renal" and "vascular" leptin resistance observed in obesity.

Modulation of paraoxonase 1 and protein N-homocysteinylation by leptin and the synthetic liver X receptor agonist T0901317 in the rat.

The adipose tissue hormone leptin and homocysteine (Hcy)-thiolactone are linked to the pathogenesis of atherosclerosis through their interactions with the anti-atherogenic enzyme paraoxonase 1 that has the ability to hydrolyze Hcy-thiolactone and minimizes protein N-homocysteinylation. Here we examined the relationships between hyperleptinemia, Hcy-thiolactonase, and protein N-homocysteinylation in rats. Hyperleptinemia was induced in adult rats by administration of leptin for 7 days (0.25 mg/kg twice daily s.c). We found that serum Hcy-thiolactonase was lower in hyperleptinemic than in control animals (-41.0%, P<0.001). Leptin administration increased the level of N-linked Hcy in plasma proteins (+92.9%, P<0.01), but had no effect on plasma total Hcy. These effects were not reproduced by pair-feeding. We also found that the synthetic liver X receptor (LXR) agonist, T0901317 (1 mg/kg per day) normalized Hcy-thiolactonase and protein N-homocysteinylation levels in leptin-treated rats. However, leptin-induced increase in plasma isoprostane levels (a marker of oxidative stress) was not normalized by T0901317. The NADPH oxidase inhibitor apocynin prevented leptin-induced increase in isoprostane levels but did not normalize Hcy-thiolactonase and protein N-homocysteinylation levels. These results suggest that the decreased capacity to metabolize Hcy-thiolactone and concomitant increase in protein N-homocysteinylation contribute to pro-atherogenic effect of chronic hyperleptinemia, independently of oxidative stress. LXR agonists normalize Hcy-thiolactonase levels and decrease protein N-homocysteinylation, especially under conditions associated with excess leptin such as metabolic syndrome.

Resistance to acute NO-mimetic and EDHF-mimetic effects of leptin in the metabolic syndrome.

AIMS: We examined mechanisms leading to the impairment of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-dependent vasorelaxation in response to acutely administered leptin in rats with the metabolic syndrome. MAIN METHODS: Effects of leptin on blood pressure and NO and cGMP in the aortic wall were studied in four groups of rats: (1) lean control, (2) obese, fed "cafeteria diet" for 3months (hyperleptinemia and hyperinsulinemia), (3) hyperleptinemia induced by administration of exogenous leptin for 8days, and (4) fructose-fed, receiving 20% fructose in the drinking water for 8weeks (hyperinsulinemia with slightly elevated leptin). KEY FINDINGS: Stimulatory effect of leptin on NO and cGMP production in the aortic wall was impaired in obese and hyperleptinemic groups but not in the fructose group. In contrast, EDHF-mimetic effect of leptin was impaired in obese and fructose-fed but not in the hyperleptinemic group. Leptin increased tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) in the aortic wall, and this effect was impaired in obese and fructose-fed animals. The EDHF-mimetic effect of leptin was abolished by phosphoinositide 3-kinase inhibitor, wortmannin, whereas its effect on NO was not. In addition, IRS-1 phosphorylation at Ser(307) and Ser(612) was enhanced in obese and fructose-fed but not in hyperleptinemic rats. SIGNIFICANCE: These results indicate that: (1) long-term hyperleptinemia induces resistance to acute vascular NO-mimetic effect of leptin in obesity/metabolic syndrome, (2) leptin stimulates EDHF in IRS-1 and PI3K-dependent manner, and this effect is impaired in obesity due to excessive serine phosphorylation of IRS-1.

Adverse effects of statins - mechanisms and consequences.

Statins inhibit 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis, which converts HMG-CoA to mevalonate. Statins lower plasma low-density lipoprotein (LDL) cholesterol by causing intracellular cholesterol depletion and upregulating the expression of LDL receptors. Apart from cholesterol, mevalonate is also the substrate for the synthesis of nonsteroid isoprenoids including farnesylpyrophosphate, geranylgeranylpyrophosphate (both attached to small GTP-binding proteins by protein prenyltransferases), coenzyme Q, dolichol, isopentenyladenosine, etc. Depletion of these isoprenoids results in so called "pleiotropic" effects of statins which are independent of cholesterol lowering. Although statins are generally well-tolerated, adverse effects may occur in some patients. These effects result from impaired protein prenylation, deficiency of coenzyme Q involved in mitochondrial electron transport and antioxidant protection, abnormal protein glycosylation due to dolichol shortage, or deficiency of selenoproteins. Myopathy is the most frequent side effect of statins and in some cases may have a form of severe rhabdomyolysis. Less common adverse effects include hepatotoxicity, peripheral neuropathy, impaired myocardial contractility and autoimmune diseases. The risk of these unfavorable effects is largely outweighed by great reduction of cardiovascular events in statin users. However, due to increasing number of patients taking statins, monitoring for any side effects, intense research to recognize their mechanisms and to identify susceptible patients, as well as rational management of these complications are mandatory to further improve safety of these excellent drugs.

Renal antioxidant enzymes and glutathione redox status in leptin-induced hypertension.

Previously, we have demonstrated that leptin increases blood pressure (BP) in the rats through two oxidative stress-dependent mechanisms: stimulation of extracellular signal-regulated kinases (ERK) by H(2)O(2) and scavenging of nitric oxide (NO) by superoxide (O(2-.)). Herein, we examined if renal glutathione system and antioxidant enzymes determine the mechanism of prohypertensive effect of leptin. Leptin administered at 0.5 mg/kg/day for 4 or 8 days increased BP and renal Na(+),K(+)-ATPase activity and reduced fractional sodium excretion; these effects were prevented by NADPH oxidase inhibitor, apocynin. Superoxide scavenger, tempol, abolished the effect of leptin on BP and renal Na(+) pump in rats receiving leptin for 8 days, whereas ERK inhibitor, PD98059, was effective in animals treated with leptin for 4 days. Leptin administered for 4 days decreased glutathione (GSH) and increased glutathione disulfide (GSSG) in the kidney. In animals receiving leptin for 8 days GSH returned to normal level, which was accompanied by up-regulation of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme of the GSH biosynthetic pathway. In addition, superoxide dismutase (SOD) activity was decreased, whereas glutathione peroxidase (GPx) was increased in rats receiving leptin for 8 days. Cotreatment with gamma-GCS inhibitor, buthionine sulfoximine (BSO), accelerated, whereas GSH precursor, N-acetylcysteine (NAC), attenuated leptin-induced changes in gamma-GCS, SOD, and GPx. In addition, coadministration of BSO changed the mechanism of BP elevation from H(2)O(2)-ERK to (O(2-.))-NO dependent in animals receiving leptin for 4 days, whereas NAC had the opposite effect in rats treated with leptin for 8 days. These results suggest that initial change in GSH redox status induces decrease in SOD/GPx ratio, which results in greater amount of (O)2-.)) versus H(2)O(2) in later phase of leptin treatment, thus shifting the mechanism of BP elevation from H(2)O(2)-ERK to (O(2-.))-NO dependent.

Transactivation of epidermal growth factor receptor in vascular and renal systems in rats with experimental hyperleptinemia: role in leptin-induced hypertension.

We examined the role of epidermal growth factor (EGF) receptor in the pathogenesis of leptin-induced hypertension in the rat. Leptin, administered in increasing doses (0.1-0.5 mg/kg/day) for 10 days, increased phosphorylation levels of non-receptor tyrosine kinase, c-Src, EGF receptor and extracellular signal-regulated kinases (ERK) in aorta and kidney, which was accompanied by the increase in plasma concentration and urinary excretion of isoprostanes and H2O2. Blood pressure and renal Na+,K+-ATPase activity were higher, whereas urinary sodium excretion was lower in animals receiving leptin. The effects of leptin on renal Na+,K+-ATPase, natriuresis and blood pressure were abolished by NADPH oxidase inhibitor, apocynin, Src kinase inhibitor, PP2, EGF receptor inhibitor, AG1478, protein farnesyltransferase inhibitor, manumycin A, and ERK inhibitor, PD98059. In contrast, inhibitors of insulin-like growth factor-1 and platelet-derived growth factor receptors, AG1024 and AG1295, respectively, only slightly reduced ERK phosphorylation and had no effect on blood pressure in rats receiving leptin. These data indicate that: (1) experimental hyperleptinemia is associated with oxidative stress and c-Src-dependent transactivation of the EGF receptor, which stimulates ERK in vascular wall and the kidney, (2) overactivity of EGF receptor-ERK pathway contributes to leptin-induced hypertension by stimulating renal Na+,K+-ATPase and reducing sodium excretion, (3) inhibitors of c-Src, EGF receptor and ERK may be considered as a novel therapy for hypertension associated with hyperleptinemia, e.g. in patients with obesity and metabolic syndrome.

Role of extracellular signal-regulated kinases (ERK) in leptin-induced hypertension.

We investigated if extracellular signal-regulated kinases (ERK) and oxidative stress are involved in the pathogenesis of arterial hypertension induced by chronic leptin administration in the rat. Leptin was administered at a dose of 0.25 mg/kg twice daily s.c. for 4 or 8 days. Blood pressure (BP) was higher in leptin-treated than in control animals from the third day of the experiment. The superoxide dismutase (SOD) mimetic, tempol, normalized BP in leptin-treated rats on days 6, 7 and 8, whereas the ERK inhibitor, PD98059, exerted a hypotensive effect on days 3 through 6. Leptin increased ERK phosphorylation level in renal and aortic tissues more markedly after 4 than after 8 days of treatment. In addition, leptin reduced urinary Na(+) excretion and increased renal Na(+),K(+)-ATPase activity, and these effects were abolished on days 4 and 8 by PD98059 and tempol, respectively. The levels of NO metabolites and cGMP were reduced in animals receiving leptin for 8 days. Markers of oxidative stress (H(2)O(2) and lipid peroxidation products) were elevated to a greater extent after 4 than after 8 days of leptin treatment. In contrast, nitrotyrosine, a marker of protein nitration by peroxynitrite, was higher in animals receiving leptin for 8 days. NADPH oxidase inhibitor, apocynin, prevented leptin's effect on BP, ERK, Na(+),K(+)-ATPase/Na(+) excretion and NO formation at all time points. SOD activity was reduced, whereas glutathione peroxidase (GPx) activity was increased in the group treated with leptin for 8 days. These data indicate that: (1) ERK, activated by oxidative stress, is involved only in the early phase of leptin-induced BP elevation, (2) the later phase of leptin-induced hypertension is characterized by excessive NO inactivation by superoxide, (3) the time-dependent shift from ERK to O(2)(-)-NO dependent mechanism may be associated with reduced SOD/GPx ratio, which favors formation of O(2)(-) instead of H(2)O(2).