Journal of Physiology and Biochemistry

Published by Springer Verlag
Online ISSN: 1877-8755
Publications
Article
In previous works we have found a mitochondrial alkaline phosphatase (AP) activity in LLC-PK1. The aim of this work has been to study the possible involvement of mitochondrial AP activity in the synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) from the substrate 25(OH)D3. Renal phenotype LLC-PK1 cells were incubated with 25(OH)D3 as substrate and treated with or without 1,25(OH)2D3, forskolin, 12-myristate-13-acetate (PMA) and 1,25(OH)2D3 in conjunction with PMA. Incubation of LLC-PK1 cells with forskolin (adenylate cyclase activator) not only stimulated the 1-hydroxylase and inhibited the 24-hydroxylase activities but also increased the mitochondrial AP activity. The addition of 1,25(OH)2D3, the main activator of 24-hydroxylase, produced a decrease of mitochondrial AP activity, a decrease of 1,25(OH)2D3 synthesis and an increase of the 24,25(OH)2D3 synthesis. Incubation with PMA, a potent activator of protein kinase C, did not produce any changes in mitochondrial AP activity, but an inhibition of 1,25(OH)2D3 and an activation of 24,25(OH)2D3 synthesis were found. Moreover, incubation of LLC-PK1 cells with PMA in conjunction with 1,25(OH)2D3 produced an additive effect in the decrease of 1,25(OH)2D3 and an increase of 24,25(OH)2D3 synthesis remaining mitochondrial AP activity as cells treated only with 1,25(OH)2D3. Our results suggest that mitochondrial AP activity could be involved as an intracellular signal in the regulation of 25(OH)D3 metabolism to the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 in renal phenotype LLC-PK1 cells through cAMP protein kinase system.
 
Article
It has been reported that some hypoparathyroid patients with magnesium deficiency showed altered responses to vitamin D treatment. In the same way, in vitro bone studies have demonstrated the existence of a decrease in the 1,25-dihydroxyvitamin D3-induced resorption in bone as a result of magnesium deficiency. These findings suggest some kind of alteration in the 1,25(OH)2D3 in bone in magnesium deficiency. In the present work, using a binding assay based on the 1,25(OH)2D3 and 3H-1,25(OH)2D3 competition for the hormone binding sites in rat calvaria homogenates, a significant decrease in the number of 1,25(OH)2D3 specific binding sites has been found in calvaria incubated in magnesium-deficient medium compared to magnesium-replete ones. Alterations in the hormone-receptor affinity were not found. These results suggest that an alteration in the 1,25(OH)2D3 action on magnesium-deficient bone could be due, at least in part, to a decrease in the number of available vitamin D receptors in bone cells.
 
Article
The aim of this work was to evaluate the effects of 24,25-dihydroxyvitamin D3, 24,25(OH)2D3, on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities in fetal rat calvaria cultures. These actions were compared with those of 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, and 25-hydroxyvitamin D3, 25(OH)D3, in similar experimental conditions. At 10 min, 30 min and at 24 h incubation time, 1,25(OH)2D3 (10(-10)M) and 25(OH)D3 (10(-7) M) produced a significant increase in AP and TRAP activities compared to control group (without vitamin D metabolites). However, 24,25(OH)2D3 (10(-7) M) only produced effects on phosphatase activities similar to those produced by 1,25(OH)2D3 and 25(OH)D3, after 24 h incubation time. These findings suggest that 1,25(OH)2D3 and 25(OH)2D3 could carry out actions in minutes (nongenomic mechanism), while 24,25(OH)2D3 needs longer periods of time to perform its biological actions (genomic mechanism).
 
Article
In order to transform cytosolic fructose-1,6-bisphosphatases (FBPase)(EC 3.1.3.11) into potential reductively-modulated chloroplast-type enzymes, we have constructed four chimeric FBPases, which display structural viability as deduced by previous modelling. In the X1-type BV1 and HL1 chimera the N-half of cytosolic sugar beet (Beta vulgaris L.) and human FBPases was fused with the C-half of the pea (Pisum sativum L.) chloroplast enzyme, which carries the cysteine-rich light regulatory sequence. In the X2-type BV2 and HL2 chimera this regulatory fragment was inserted in the corresponding site of the sugar beet cytosolic and human enzymes. Like the plant cytosolic FBPases, the chimeric enzymes show a low rise of activity by dithiothreitol. Both BV1 and BV2, but not HL1 and HL2, display a negligible activation by Trx f, but neither of them by Trx m. Antibodies raised against the pea chloroplast enzyme showed a positive reaction against the four chimeric FBPases and the human enzyme, but not against the sugar beet one. The four chimera display typical kinetics of cytosolic FBPases, with Km values in the 40-140 microM range. We conclude the existence of a structural capacity of cytosolic FBPases for incorporating the redox regulatory cluster of the chloroplast enzyme. However, the ability of these chimeric FBPases for an in vitro redox regulation seems to be scarce, limiting their use from a biotechnology standpoint in in vivo regulation of sugar metabolism.
 
a Reverse transcription-polymerase chain reaction (PCR) amplification of sodium-calcium exchanger (NCX) in different tissues. −RT control PCR without reverse transcriptase for NCX, TSM tracheal smooth muscle, M 100 bp ladder DNA marker. Representative of several assays performed. b Sequence of the alternatively spliced region of NCX1 in guinea pig TSM and comparison with the human tracheae and rabbit kidney. Numbering is according to the guinea pig sequence. Differences in nucleotide sequences with respect to guinea pig are in bold, and box highlights important amino acid substitutions between guinea pig and human NCX 
Article
The sodium-calcium exchanger (NCX) plays a major role in the regulation of cytosolic Ca(2+) in muscle cells. In this work, we performed force experiments to explore the role of NCX during contraction and relaxation of Cch-stimulated guinea pig tracheal smooth muscle strips. This tissue showed low sensitivity to NCX inhibitor KB-R7943 (IC50, 57 +/- 2 microM), although a complete relaxation was obtained by NCX inhibition at 100 microM. Interestingly, relaxation after washing the agonist was prolonged in the absence of external Na(+), whereas washing without Na(+) and in the presence of KB-R7943 resembled control conditions with physiological solution. Altogether, this suggests the reversal of NCX to a Ca(2+) influx mode by the manipulation on the Na(+) gradient, which can be inhibited by KB-R7943. In order to understand the low sensitivity to KB-R7943, we studied the molecular aspects of the NCX expressed in this tissue and found that the isoform of NCX expressed is 1.3, similar to that described in human tracheal smooth muscle. Sequencing revealed that amino acid 19 in exon B is phenylalanine, whereas in its human counterpart is leucine, and that the first amino acid after exon D is aspartate instead of glutamate in humans. Results herein presented are discussed in term of their possible functional implications in the exchanger activity and thus in airway physiology.
 
Article
Adolescence constitutes a period of nutritional vulnerability due to increased dietary requirements for growth and development and special dietary habits. A pilot trial was performed to evaluate the dietary calcium utilization among a sample of Spanish boys on their usual diets, in which the calcium intake and consumption of dairy products were as well examined. Nutrient and food intake was recorded using a 24-h dietary recall and a 2-d food consumption record for 21 subjects aged 11-14 years. Dietary calcium utilization was assessed by means of calcium intake in food and calcium output in faeces and urine as measured by flame atomic absorption spectrophotometry. Overall intake of dairy products was 399.3 +/- 22.1 g/d, and the single most consumed item was milk (72% of the total). An inverse relationship was found between dairy product consumption at breakfast (55% of the total) and BMI (p = 0.016, r = -0.5168). Dairy products contributed the majority of dietary calcium (66.9%). Mean calcium intake was 881.7 +/- 39.9 mg/d, 88% of the recommended value for Spanish adolescents. Net calcium absorption (271.7 +/- 51.7 mg/d) and retention (170.6 +/- 50.9 mg/d) seemed not to be sufficient to meet growth demands during puberty. The results shown that adolescents of the study absorbed 31% of dietary calcium and retained nearly 20% of the total intake, but dietary calcium intake and consumption of dairy products failed to meet recommended values. Optimizing calcium intake is of crucial importance among adolescents, to maximize calcium retention and to help prevent osteoporosis in later life.
 
Article
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) with unknown etiology and pathogenesis. A local autoimmune process involving activation of autoreactive T cells against CNS protein components is likely crucial in the development of MS lesions. Myelin-reactive T cells are believed to be primed in the periphery during infections by antigens of bacterial or viral origin via molecular mimicry, a postulated mechanism that might account for the trigger of an autoimmune response on the basis of sequence homology between foreign and self determinants. Immune responses to heat shock proteins (hsp) have been implicated in the initiation or progression of a number of autoimmune diseases. Hsp may function as immunodominant targets during the immune response evoked by pathogens, and theoretically a cross-reactive response to sequences shared by these immunogens and autoantigens in the CNS may contribute to the pathogenesis of MS. We examined the immune response of peripheral blood mononuclear cells (PBMNc) from MS patients and healthy subjects elicited by peptides derived from hsp60 containing a common structural motif ("2-6-11" motif) already described, which is also present in CNS putative antigens. This structural pattern consists of an apolar residue or Lys at position 2, Pro always at position 6, and Glu, Asp or Lys at residue 11. Results reported here are indicative of maturation of peripheral blood monocytes towards a differentiated CD14(+)CD16(+)DR(+) cell and release of pro-inflammatory cytokines consistent with a Th1-like pattern. These are typical features exhibited by immune cells implicated in autoimmune responses.
 
(a) Structure of glycyrrhetinic acid GA, a HSD1 inhibitor and of corticosterone (b), substrate of HSD1.
Effect of GA on HSD1 (a) and PEPCK activity (b) in control and stressed animals. Results represent mean ± SEM of 8 different experiments performed in duplicate. ### p<0.001; ## p<0.01 vs. Control group. ***p<0.001 vs. HCl group. GA, glycyrrhetinic acid; HSD1, 11‚-hydroxysteroid dehydrogenase 1; PEPCK, phosphoenolpyruvate carboxykinase. 
Article
11 beta-hydroxysteroid dehydrogenase (HSDs) enzymes regulate the activity of glucocorticoids in target organs. HSD1, one of the two existing isoforms, locates mainly in CNS, liver and adipose tissue. HSD1 is involved in the pathogenesis of diseases such as obesity, insulin resistance, arterial hypertension and the Metabolic Syndrome. The stress produced by HCl overload triggers metabolic acidosis and increases liver HSD1 activity associated with increased phosphoenolpyruvate carboxykinase, a regulatory enzyme of gluconeogenesis that is activated by glucocorticoids, with increased glycaemia and glycogen breakdown. The aim of this study was to analyze whether the metabolic modifications triggered by HCl stress are due to increased liver HSD1 activity. Glycyrrhetinic acid, a potent HDS inhibitor, was administered subcutaneously (20 mg/ml) to stressed and unstressed four months old maleSprague Dawley rats to investigate changes in liver HSD1, phosphoenolpyruvate carboxykinase (PECPK) and glycogen phosphorylase activities and plasma glucose levels. It was observed that all these parameters increased in stressed animals, but that treatment with glycyrrhetinic acid significantly reduced their levels. In conclusion, our results demonstrate the involvement of HSD1 in stress induced carbohydrate disturbances and could contribute to the impact of HSD1 inhibitors on carbohydrate metabolism and its relevance in the study of Metabolic Syndrome Disorder and non insulin-dependent diabetes mellitus.
 
Article
The present study was undertaken to investigate the effect of the new formyl peptide receptor 2/lipoxin A4 receptor agonist BML-111 on acetaminophen (APAP)-induced liver injury in mice and explore its possible mechanism(s). Male Swiss albino mice were intraperitoneally injected with BML-111 (1 mg/kg) twice daily for five consecutive days prior to a single intraperitoneal injection of APAP (500 mg/kg). Results have shown that APAP injection caused liver damage as indicated by significant increase in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Liver histopathological examination revealed marked necrosis and inflammation. Additionally, APAP decreased activities of hepatic glutathione (GSH) and superoxide dismutase (SOD) with significant increase in the hepatic malondialdehyde (MDA) content. Furthermore, APAP increased serum nitrite/nitrate (NO2 (-)/NO3 (-) ) level and hepatic tumor necrosis factor alpha (TNF-α). Pretreatment with BML-111 significantly reversed all APAP-induced pathological changes. BML-111 prevented the increase of AST, ALT, and ALP. Also, BML-111 markedly attenuated APAP-induced necrosis and inflammation. It decreased MDA with increase in SOD and GSH. Importantly, BML-111 decreased NO2 (-)/NO3 (-) level and TNF-α. These findings suggest that BML-111 has hepatoprotective effects against APAP-induced liver injury in mice. Its protective effect may be attributed to its ability to counteract the inflammatory ROS generation and regulate cytokine effects.
 
Article
To explore if protective effect of melatonin on oxidative stress induced by okadaic acid, an inhibitor of protein phosphatases PP1 and PP2A, is mediated by membrane receptors subtype mt1, we used an in vitro model with N1E-115 neuroblastoma cells. We demonstrated that exposure of cells to 50 nM okadaic acid for 2 h induces a reduction in the activity of antioxidative enzymes, and an increase of lipid peroxidation products, while melatonin prevents the effect of okadaic acid. On the other hand, the presence of luzindole, 20 min before adding melatonin, did not cause changes on the effect of the melatonin on oxidative stress. These results seem to indicate that protective effect of melatonin is not mediated by mt1 receptors.
 
Mean serum levels of hepatic marker enzymes in Wistar rats 
activities of enzymatic antioxidants and mean levels of nonenzymatic antioxidants and malondialdehyde in hepatic tissue samples from Wistar rats 
Article
Hypercholesterolemia and oxidative stress are known to accelerate coronary artery disease and progression of atherosclerotic lesions. In the present study, an attempt was made to evaluate the putative antihypercholesterolemic and antioxidative effects of an ethanolic extract of the oyster mushroom (Pleurotus ostreatus) and chrysin, one of its major components, in hypercholesterolemic rats. Hypercholesterolemia was induced in rats by a single intraperitoneal injection of Triton WR-1339 (300 mg/kg body weight (b.wt.)), which resulted in persistently elevated blood/serum levels of glucose, lipid profile parameters (total cholesterol, triglycerides, low-density lipoprotein-, and very low-density lipoprotein-cholesterol), and of hepatic marker enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase). In addition, lowered mean activities of hepatic antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) and lowered mean levels of nonenzymatic antioxidants (reduced glutathione, vitamin C, and vitamin E) were observed. Oral administration of the mushroom extract (500 mg/kg b.wt.) and chrysin (200 mg/kg b.wt.) to hypercholesterolemic rats for 7 days resulted in a significant decrease in mean blood/serum levels of glucose, lipid profile parameters, and hepatic marker enzymes and a concomitant increase in enzymatic and nonenzymatic antioxidant parameters. The hypercholesterolemia-ameliorating effect was more pronounced in chrysin-treated rats than in extract-treated rats, being almost as effective as that of the standard lipid-lowering drug, lovastatin (10 mg/kg b.wt.). These results suggest that chrysin, a major component of the oyster mushroom extract, may protect against the hypercholesterolemia and elevated serum hepatic marker enzyme levels induced in rats injected with Triton WR-1339.
 
Article
The main aim of this study was to examine the hypothesis that creatine (Cr) feeding enhances myocellular glycogen storage in humans undergoing carbohydrate loading. Twenty trained male subjects were randomly assigned to have their diets supplemented daily with 252 g of glucose polymer (GP) and either 21 g of Cr (CR-GP, n = 10) or placebo (PL-GP, n = 10) for 5 days. Changes in resting myocellular glycogen and phosphocreatine (PCr) were determined with Magnetic Resonance Spectroscopy (13C- and 31P-MRS, respectively). After CR-GP, the levels of intramyocellular glycogen increased from 147 +/- 13 (standard error) mmol x (kg wet weight(-1)) to 172 +/- 13 m mol x (kg wet weight)(-1), while it increased from 134 +/- 17 mmol x (kg wet weight)(-) to 182 +/- 17 mmol x (kg wet weight)(-1) after PL-GP; the increments in intramyocellular glycogen concentrations were not statistically different. The increment in the PCr/ATP ratio after CR-GP (+ 0.20 +/- 0.12) was significantly different compared to PL-GP (- 0.34 +/- 0.16) (p < 0.05). The present results do not support the hypothesis that Cr loading increases muscle glycogen storage.
 
Article
The current study was designed to explore the potential involvement of miR-155 in the pathogenesis of diabetes complications. Male rats were divided into control and diabetic groups (n = 6). Type 2 diabetes was induced by a single-dose injection of nicotinamide (110 mg/kg; intraperitoneal (i.p.)), 15 min before injection of streptozotocin (STZ; 50 mg/kg; i.p.) in 12-h fasted rats. Two months after induction of diabetes, the rats were sacrificed for subsequent measurements. The nuclear factor kappa B (NF-κB) activity was higher in diabetic peripheral blood mononuclear cells (PBMCs), aorta, heart, kidney, liver, and sciatic nerve, than the control counterparts. Also, apoptosis rate was increased in these tissues, except the aorta. NF-κB messenger RNA (mRNA) expression level was higher in the kidney, heart, PBMCs, and sciatic nerve of diabetic rats than their control counterparts. Except the liver, the miR-155 expression level was significantly decreased in diabetic kidney, heart, aorta, PBMCs, and sciatic nerve versus the controls. Moreover, the expression of miR-155 was negatively correlated with NF-κB activity and apoptosis rate. These results suggest that changes in the expression of miR-155 may participate in the pathogenesis of diabetes-related complications, but causal relationship between miR-155 dysregulation and diabetic complications is unknown.
 
Article
Aim To investigate the association of matrix metalloproteinase-3 (MMP-3) polymorphism with susceptibility to coronary artery stenosis (CAS) and the number of diseased vessels in patients with type 2 diabetes mellitus (T2DM). Methods The study population comprised 618 unrelated Iranian individual subjects, including 305 angiographically documented CAS patients with T2DM and 313 control subjects with T2DM. MMP3 genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism. Results Significant differences between cases and controls were observed for MMP3 genotype frequencies (p < 0.01). The 6A allele was high frequently seen in the disease group, compared with the control group (64.75 vs. 56.24%, 6A/6A + 5A/6A vs. 5A/5A, p < 0.05). The association of this polymorphism with the severity of stenosis were also evaluated which according to results distribution of MMP3 genotypes were not significantly different as compared with the severity of stenosis (p > 0.05). Conclusions Frequency of the 6A allele of the human MMP3 gene is an independent risk factor for CAS in the Iranian T2DM studied.
 
Article
In aging liver oxidative stress increases due to the decrease in antioxidant bio-molecules such as estrogens which can be modified by hormonal replacement therapy (HRT). With this in mind, we hypothesized that age-related decline in steroidogenesis may be associated with the impairment of the antioxidant defense cells in liver, the increase in lipid peroxidation, hepatic dysfunction and histological changes; estrogens prevent all these changes induced by aging. 17beta-estradiol treatment was initiated in 12 month-old Wistar rats, and continued until 18 months of age. Our results showed that 17beta-estradiol (E2) level in the serum of the aged untreated rats was reduced by -32% in 18 month-old rats compared to the young animals (4-month-old). The superoxide dismutase (SOD), catalase (CAT), and gluthatione peroxidase (GPX) activities were reduced by -47, -46, and -29% respectively in old rat liver. In addition, the TBARs in liver and hepatic dysfunction parameters in plasma such as gamma-glutamyl transferase (GGT), phosphatase alkalin (PAL) as well as bilirubin level increased significantly in old rats, and histological changes were investigated. In E2-treated rats, protective effects were observed. Indeed, 17beta-estradiol attenuates all changes induced by aging. The 17beta-estradiol level was higher in old E2-treated rats compared to the control rats. Moreover, the SOD, CAT and GPX activities were higher by +28, +15, and +11% respectively. This anti-aging effect of estrogens was clarified by a lower level of lipid peroxidation and liver dysfunction parameters as well as by histological observation.
 
Article
Increased intracelullar hormone concentration levels have been shown to be the cause of several endocrine-related cancers including breast, prostate, endometrial, ovarian, cervix, testicular, thyroid, and osteosarcoma. Deregulated expression of steroidogenic enzymes in these tumors seems to be the source of a positive balance in active steroids that bind to the corresponding nuclear receptor, thus ultimately stimulating cell proliferation. Among these enzymes, 17β-hydroxysteroid dehydrogenases catalyze the interconversion between 17-ketosteroids and 17-hydroxysteroids on the last steps of sex hormones biosynthesis and metabolism. To date, 14 isoforms have been identified in vertebrates although only 13 are present in humans. Development and clinical evaluation of specific inhibitors to block their activity is currently under progress especially against the best characterized members 1 to 5. Selectivity and potency of these drugs constitute the main challenge in this new approach to cancer and steroid-dependent diseases treatment at the "pre-receptor level". Here we review the current state of knowledge regarding the deregulation of the expression of some of these enzymes in endocrine-related tumors.
 
Regression coefficients, SEM and R 2 showing the association between body fat mass percentage and inflammatory plasma markers in the whole sample Controlling by age, gender, pubertal status and physical activity 
Regression coefficients, SEM and R 2 showing the association between body fat mass percentage and inflammatory markers according to the -174G/C polymorphism of the IL6 gene. Controlling by age, gender, pubertal status and physical activity 
Article
During the last decades, the prevalence of obesity has increased rapidly among young people. A polymorphism in the promoter region of the IL6 gene (-174G/C), has been previously reported to be involved in obesity and metabolic syndrome development. Therefore, the aim of the study was to examine whether the IL6-174G/C polymorphism influence the association of body fat with low-grade inflammatory markers and blood lipids and lipoproteins in Spanish adolescents. 504 Spanish adolescents participating in the AVENA study were genotyped for the-174G/C polymorphism of the IL6 gene. Anthropometric and body composition measurements were taken and blood samples were collected for plasma molecules determinations. No differences between genotypes were observed in anthropometric values, body composition measurements and plasma markers concentration. Physical activity level differ between genotypes with subjects carrying the C allele of the polymorphism being significantly (p<0.05) more active than GG subjects. The association between body fat mass and plasma glucose was influenced by the -174G/C polymorphism of the IL6 gene. Subjects carrying the C allele of the mutation seem to have higher values of lipoprotein (a) and C-reactive protein as their percentage of body fat mass increase. Our results suggest that this promoter polymorphism influences the association between adiposity and some plasma markers.
 
Schematic representation showing the experimental protocols used for the study. a Unprotected ischemia-reperfusion (control). b Protection with PPC. c Protection with E2 perfusion. 
Effects of PPC, E2 perfusion, and E2 substitution on the recovery of the heart contractility and coronary vascular dynamics. a Post-ischemic recovery in LV contractility, DPmax, and b LVEDP. c Recovery in the coronary vascular dynamics, CF, and d CVR after ischemia and PPC. The data were collected at 30 min reperfusion and were presented as means ± SEM. 
Article
The role of pacing postconditioning (PPC) in the heart protection against ischemia-reperfusion injury is not completely understood. The aim of this study was to investigated if 17-β-estradiol (estrogen, E2), endogenous atrial natriuretic peptide (ANP), endogenous brain natriuretic peptide (BNP), and tumor necrosis factor-alpha (TNF-α) are involved in PPC-mediated protection. Langendorff perfused female Wistar rat hearts were used for this study. Hearts challenged with regional ischemia for 30 min subjected to no further treatment served as a control. The PPC protocol was 3 cycles of 30 s pacing alternated between the right atrium and left ventricle (LV). Protection was assessed by recovery of LV contractility and coronary vascular-hemodynamics. Ischemia induced a significant (P < 0.05) deterioration in the heart function compared with baseline data. PPC alone or in combination with short-term E2 treatment (E2 infusion at the beginning of reperfusion) significantly (P < 0.05) improved the heart functions. Short-term E2 treatment post-ischemically afforded protection similar to that of PPC. However, long-term E2 substitution for 6 weeks completely attenuated the protective effects of PPC. Although no changes were noted in endogenous ANP levels, PPC significantly increased BNP expression level and decreased TNF-α in the cardiomyocyte lysate and coronary effluent compared to ischemia and controls. Our data suggested a protective role for short-term E2 treatment similar to that of PPC mediated by a pathway recruiting BNP and downregulating TNF-α. Our study further suggested a bad influence for long-term E2 substitution on the heart as it completely abrogated the protective effects of PPC.
 
Article
The antiprogesterone RU486 injected on proestrus to cyclic rats advances the preovulatory surge of LH, resulting in a 3-day estrous cycle. To ascertain whether proestrous progesterone secretion regulates ovulation by synchronizing the functions of the ovary, the pituitary and the hypothalamus, in this study the effects of RU486 (4 mg/0.2 ml oil/s.c.) at 09:00 h on proestrus (day 1), on LHRH secretion into the pituitary stalk blood vessels and on peripheral plasma concentrations of LH and 17beta-estradiol at 10:00 h and at 18:00 h on each day of the estrous cycle, have been investigated. Control rats present the expected surges of LHRH and LH at 18:00 h on day 1 and a second increase in LHRH but no LH secretion was found in the afternoon of day 2. RU486 decreases both LHRH and LH surges in the afternoon of day 1, while it increases plasma concentrations of LHRH and LH in the morning of day 2. During the rest of the estrous cycle RU486-injected rats show high basal secretion of LH in comparison to controls, and at 18:00 h of day 4 advanced preovulatory surges of both LHRH and LH are present in RU486-injected rats. In relation to 17beta-estradiol concentrations, RU486 injection increases those on day 3 and induces an advanced preovulatory surge of 17beta-estradiol in the morning of day 4. These results show that, in the 4-day cyclic rat, the 24 hours-shortening of the estrous cycle induced by the antiprogesterone RU486 is due to the enhanced secretion of estrogen during the days of metestrus and diestrus, which stimulates the release of a surge of LHRH in the afternoon of diestrus and this, in turn, induces the advanced preovulatory surge of LH.
 
Article
The plasma estradiol-17beta levels (E2-17beta) and gonadosomatic index (GSI) of female tench (Tinca tinca L.) reared outdoors and indoors were studied from December 1996 to April 1997. Water temperature and daylight were gradually increased (from 10 to 25 degrees C and from 9 to 16 hours per day, respectively) in the indoor group. Animals reared in natural conditions had similar plasma levels throughout the experiment but animals under controlled conditions experienced an increase in these levels in March in relation to previous months (4.81+/-0.73 ng/ml in March vs. 0.29+/-0.03 ng/ml and 0.99+/-0.31 ng/ml in January and February, respectively; P < 0.05) and in relation to animals reared outdoors (4.81+/-0.73 ng/ml vs. 0.47+/-0.19 ng/ml, respectively; P < 0.05). In April, a sharp decrease in estradiol-17beta in these animals was observed, probably associated with the end of vitellogenesis. GSI increased in both groups through the experiment and indoor animals showed greater values in March vs outdoor fishes (7.57+/-2.18 vs. 4.05+/-0.63; P < 0.05). No significant correlation was found between E2-17beta and GSI, (r = 0.35). It is concluded that manipulation of the photoperiod and temperature can modify gonadal development in tench, and this is similar to the situation in other Teleostei species. This research is the first step towards achieving, out of season, spawning in tench.
 
Plasma IL-6 ( a ) and IL-18 ( b ) concentrations ( n =153), according to the distribution of waist circumference (centimeters) into tertiles, and IL-6 ( c ) and IL-18 ( d ) according to the 
Multiple linear regression analyses with IL-6 concentrations (pg/mL) as a predictor of total cholesterol, TyG index, CRP, and C3 complement adjusted by number of cigarettes smoked per day ( n =153) 
Multiple linear regression analyses showing the independent contributions of variables of studied domains to the variation of the plasma IL-6 and IL-18 concentrations
Article
IntroductionInflammation is a physiological defense response to injury and foreign substances, in order to maintain body homeostasis [27]. However, it can also be triggered by disorders, such as obesity or diabetes, in metabolically active tissues [4].Obesity and excessive body fat content are associated with increased circulating levels of proinflammatory cytokines [47, 18], supporting a link between chronic low-grade inflammation and diseases such as diabetes and insulin resistance [41], dyslipidemia [18], and, consequently, metabolic syndrome (MetS) features [36]. In addition, previous studies have reported that weight loss leads to a reduction of these proinflammatory molecules [17], making the cytokines an interesting biomarker to follow changes associated to cardiometabolic risk and obesity management. On the other hand, it has been postulated that inflammation could trigger obesity [28]. Thaler and Schwartz (2010) proposed that inflammation in hypothalamic cells, caused by exces ...
 
- Study Design and experimental protocol 
-Plasma interleukin-6 (IL-6), IL-6sR and gp130 levels in monozygotic twins after exercise (blue circles) or after resting (red circles)
Article
Increased interleukin-6 (IL-6) plasma levels have been described to occur during physical exercise. A relative reduction in energy intake after physical activity has also been reported after exercise, indicating a possible involvement of IL-6 as an anorexigenic factor. Given the possible effect of interleukins on appetite, we assessed whether a controlled physical activity bout is related with changes in IL-6, IL-6 soluble receptor (IL-6sR), gp130 and interleukin-18 (IL-18) plasma levels, as well as their relation with post-exercise energy intake. A co-twin intervention study was carried out with five young male monozygotic twin pairs. One co-twin performed 45 min of submaximal exercise on a treadmill near the anaerobic threshold ending with 7 min at 90 % VO(2) max, while his co-twin remained non-active. Ad libitum energy intake was tested through a carbohydrate-rich meal test. Venous blood samples were drawn at baseline, immediately after exercise and after the meal ingestion. Plasma concentrations of IL-6, IL-6sR, gp130 and IL-18 were measured via ELISA. IL-6 plasma levels increased after physical activity bout (2.6-fold change; p = 0.04). A less marked trend, although still significant, was observed for plasma levels of IL-6sR and gp130. Plasma levels of IL-18 did not significantly change during exercise. The twins who exercised exhibited significantly lower energy intake (181 versus 1,195 kcal; p = 0.04), compared to the co-twins who remained resting. The present study in monozygotic twins shows increased IL-6 plasma levels after acute physical exercise with a significant reduction in energy intake, supporting a linkage between IL-6 and acute post-exercise eating behaviour.
 
Article
Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.
 
Article
The 26S proteasome is a key component of the ubiquitin-proteasome system, a process responsible for the majority of cellular protein degradation. The function of the proteasomal ubiquitin receptor hRpn13, a component of the 26S proteasome, is not completely understood. To investigate the role of hRpn13 in the ubiquitin-proteasome system in osteoblasts, the effects of suppressing and overexpressing the hRpn13 gene on proliferation, differentiation, and function of human osteoblast-like MG63 cells were examined. After knockdown of hRpn13 by small interfering RNA, changes in osteoblast proliferation were evaluated by methyl-thiazolyl-tetrazolium assay. There was an increase in markers for osteoblast proliferation, specifically alkaline phosphatase activity, and elevated protein levels of osteocalcin, proliferating cell nuclear antigen (PCNA), and ubiquitin. Furthermore, hRpn13 knockdown also resulted in a decrease in the ratio between the gene expressions of RANKL and OPG, key players in the pathogenesis of bone diseases that influence the normal balance between bone formation and resorption. In contrast, overexpression of hRpn13 inhibited the proliferation of MG63 cells, and decreased alkaline phosphatase activity as well as protein levels of osteocalcin, PCNA, and ubiquitin while the ratio of RANKL to OPG expression increased. To confirm the function of hRpn13 in the ubiquitin-proteasome pathway, osteoblast proliferation enhancement and ubiquitin accumulation after hRpn2 knockdown was assessed. The results suggest that overexpression of hRpn13 negatively influences proliferation and osteogenic differentiation in MG63 cells. The evidence implies that hRpn13 modulates the influence of osteoblasts on osteoclasts by controlling the stability of regulatory proteins in osteoblasts. In summary, overexpression of hRpn13 promoted the activity of the ubiquitin-proteasome system.
 
Article
The 26S proteasome is a key component of the ubiquitin-proteasome system, a process responsible for the majority of cellular protein degradation. The function of the proteasomal ubiquitin receptor hRpn13, a component of the 26S proteasome, is not completely understood. To investigate the role of hRpn13 in the ubiquitin-proteasome system in osteoblasts, the effects of suppressing and overexpressing the hRpn13 gene on proliferation, differentiation, and function of human osteoblast-like MG63 cells were examined. After knockdown of hRpn13 by small interfering RNA, changes in osteoblast proliferation were evaluated by methyl-thiazolyl-tetrazolium assay. There was an increase in markers for osteoblast proliferation, specifically alkaline phosphatase activity, and elevated protein levels of osteocalcin, proliferating cell nuclear antigen (PCNA), and ubiquitin. Furthermore, hRpn13 knockdown also resulted in a decrease in the ratio between the gene expressions of RANKL and OPG, key players in the pathogenesis of bone diseases that influence the normal balance between bone formation and resorption. In contrast, overexpression of hRpn13 inhibited the proliferation of MG63 cells, and decreased alkaline phosphatase activity as well as protein levels of osteocalcin, PCNA, and ubiquitin while the ratio of RANKL to OPG expression increased. To confirm the function of hRpn13 in the ubiquitin-proteasome pathway, osteoblast proliferation enhancement and ubiquitin accumulation after hRpn2 knockdown was assessed. The results suggest that overexpression of hRpn13 negatively influences proliferation and osteogenic differentiation in MG63 cells. The evidence implies that hRpn13 modulates the influence of osteoblasts on osteoclasts by controlling the stability of regulatory proteins in osteoblasts. In summary, overexpression of hRpn13 promoted the activity of the ubiquitin-proteasome system.
 
Article
The aim of the eurrent study is to investigate the therapeutic and preventive effects of 1α, 25dihydroxyvitaminD3 (1,25 (OH)2 D3) andAjuga iva (AI) extraet on diabetes toxicity in rats testes. Thus diabetic rats were treated with 1α, 25dihydroxyvitaminD3 orAjuga iva extract as both therapeutie and preventive treatments on diabetes toxicity in rats testes. Our results showed that diabetes indueed a decrease in testosterone and 17β-estradiol levels in testes and plasma. Besides, a fall in testicular antioxidant capacity appeared by a deerease in both antioxidant (superoxide dismutase (SOD), eatalase (CAT) and glutathione peroxidase (GPx) activities) and non-enzymatic antioxidant (copper (Cu), magnesium (Mg) and iron (Fe) levels). All theses changes enhanced testicular toxicity (inerease in testicular aspartate amino transaminase (AST), alanine amino transaminase (ALT), laetate dehydrogenase (LDH) activities and the lipid peroxidation and triglyeeride (TG) levels). In addition, a decrease in testicular total cholesterol (TCh) level was observed in diabetic rats testes. All the ehanges lead to a decrease in the total number and mobility of epididymal spermatozoa. The administration of 1α,25dihydroxyvitaminD3 andAjuga iva extract three weeks before and after diabetes induetion interfered and prevented diabetes toxicity in the reproduetive system. 1,25 (OH)2 D3 andAjuga iva extract blunted all changes observed in diabetic rats. To sum up, the data suggested that 1,25 (OH)2 D3 andAjuga iva extract have a protective effect on alloxan-induced damage in reproductive system by enhancing the testosterone and 17β-estradiol levels, consequenty protecting from oxidative stress, cellular toxicity and maintaining the number and motility of spermatozoids.
 
Article
Colorectal cancer is a major health problem worldwide. The aim of this study was to determine a role of microRNA-21 (miRNA-21) in colorectal cancer (CRC) and to elucidate miRNA-21 regulation of hTERT by phosphatase and tensin homologue (PTEN). Protein and mRNA samples were extracted with 30 CRC samples and one CRC cell lines. The expression of miRNA-21, hTERT, PTEN in CRC tissues and CRC cell lines was measured by real-time fluorescent quantitative PCR, Western blotting. Cell viability was detected by MTT and cell cycle assay. In this study, we show that the expression of miRNA-21 was overexpressed in CRC tissue and CRC cell lines compared with the control group. The effects of miRNA-21 were then assessed in MTT assays through in vitro transfection with a miRNA-21 mimic or inhibitor. PTEN has been identified as a target gene of miRNA-21 in CRC cell lines. Moreover, Western blot and qRT-PCR analyses revealed that miRNA-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/ERK1/2. In addition, Western blot analyses confirmed that an inverse correlation between PTEN and hTERT levels of high miRNA-21 RNA-expressing CRC tissues and cell lines. Finally, these data indicate that miRNA-21 regulates hTERT expression via the PTEN/ERK1/2 signaling pathway, therefore controlling CRC cell line growth. MiRNA-21 may serve as a novel therapeutic target in CRC.
 
Article
We have previously reported that a serine/threonine protein kinase, Cot/Tpl2, is a negative regulator of Th1-type immunity through inhibiting IL-12 expression in antigen presenting cells (APCs) stimulated by Toll-like receptor (TLR) ligands. We here show that Cot/Tpl2(-/-) macrophages produce significantly less IL-23, an important regulator of Th17-type response, than the wild-type counterparts in response to lipopolysaccharide (LPS), which is a ligand for TLR4. The decreased IL-23 production in Cot/Tpl2(-/-) macrophages is, at least partly, regulated at the transcriptional level, as the LPS-mediated IL-23 p19 mRNA induction was significantly less in Cot/Tpl2(-/-) macrophages. Chemical inhibition of extracellular signal-regulated kinase (ERK) activity similarly inhibited IL-23 expression in LPS-stimulated wild-type macrophages. As Cot/Tpl2 is an essential upstream component of the ERK activation pathway of LPS, it is suggested that Cot/Tpl2 positively regulates IL-23 expression through ERK activation. These results indicate that Cot/Tpl2 may be involved in balancing Th1/Th17 differentiation by regulating the expression ratio of IL-12 and IL-23 in APCs.
 
, HU significantly (P<0.05) increased the expression of MLC (b) but decreased the expression of phosphorylated MLC (p-MLC) at Ser 19 (c). Thus, when normalized by total MLC, as shown in d, the level of p-MLC at Ser 19 was further decreased by HU (P<0.01), indicating less phosphorylative effect of ROCK on MLC at Ser 19 .
Representative Western blots and bar graphs depicting the protein expression of total p65 and IκBα (a), nuclear p65 (b), and iNOS (c) from CON and HU rats. Bar graph data are mean±SE of protein expression assayed in at least duplicate, quantified by
Statistical bar graphs depicting the optical density at λ= 540 nm representing the relative nitrate and nitrite content in femoral arteries from CON, CON + Y-27632, CON + Y-27632 + L-NAME + NAAN, HU, HU + Y-27632, and HU + Y-27632 + L-NAME + NAAN group. * P<0.05, ** P<0.01 vs. corresponding CON; ## P<0.01 vs. absence of Y-27632 or presence of Y-27632 + L-NAME + NAAN (column 5 vs. column 4 or 6); † † P<0.01 vs. absence of Y-27632 (column 3 vs. column 1), n=6 in each group
Article
Previous studies have demonstrated inconsistent roles of Rho kinase (ROCK) in the decreased vasoconstriction of rat hindquarter vessels induced by hindlimb unweighting (HU). The present study was designed to determine the unclear role of ROCK in the mediation of HU-induced decreased femoral arterial vasoconstriction. 28-day HU rat was adopted as the animal model. With or without Y-27632, a ROCK inhibitor, isometric force of femoral artery was measured. The expression of ROCK and its effects on downstream targets were also examined. Results showed that (1) HU caused a significant decrease of the phenylephrine (PE)-evoked and potassium chloride (KCl)-evoked femoral arterial vasoconstriction (P < 0.05), confirming the functional findings by previous studies. (2) Inhibition of ROCK with Y-27632 produced an equal reduction of the vasoconstriction in CON and HU. (3) HU significantly decreased ROCK II expression and the effects of ROCK on myosin light-chain phosphatase (MLCP) and MLC (P < 0.05), but increased p65 nuclear translocation (P < 0.05) and inducible nitric oxide synthase (iNOS) expression (P < 0.05). (4) HU significantly (P < 0.05) increased NO production in femoral arteries, with Y-27632 significantly (P < 0.01) amplifying this effect. These findings have revealed that 28-day HU reduced the expression and effects of ROCK on downstream targets both directly (MLCP and MLC) and possibly indirectly (NF-κB/iNOS/NO pathway) related to vasoconstriction in femoral arteries.
 
Mean arterial pressure, left ventricular pressure, and heart rate values (mean±SEM) in acute and chronic phases of Goldblatt hypertension Phase Acute Chronic 
Apelin receptor mRNA (a) and protein (b) expression (APJ/β-actin ratio) in the myocardium of sham and 2K1C rats after 4 weeks (acute) and 16 weeks (chronic). n=7 in each group; *P<0.05 and **P<0.01 between 2K1C and related sham values 
Article
Experimental and clinical evidences suggest that apelin and its receptor APJ are involved in the pathogenesis of cardiovascular complications. However, the role of apelin/APJ in hypertension is not sufficiently understood. Because chronic kidney diseases lead to hypertension and cardiac failure, we investigated the changes in apelin receptor gene expression in the myocardium and aorta of rat models of kidney disease hypertension. Two-kidney, one-clip (2K1C) hypertension was produced by placing a clip around the renal artery. Four and 16 weeks later, blood pressure, left ventricular end-diastolic pressure (LVEDP), serum apelin, and angiotensin II were measured. The messenger RNA (mRNA) and protein of APJ were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Chronic hypertensive rats had approximately 10 times higher LVEDP (P < 0.001). 2K1C decreased serum apelin from 220 ± 11 to 170 ± 10 pg/mL in 16 weeks (P < 0.05). The mRNA expression of APJ significantly decreased in the heart and aorta at 4 weeks. At 16 weeks, the reduction was not significant in the heart but was significant in the aorta. At 4 weeks, the expression of the APJ protein significantly decreased in the heart but not in the aorta. At 16 weeks, APJ protein was significantly decreased only in the aorta. Reduction of serum apelin and downregulation of apelin receptors in both the heart and aorta may play a role in the pathophysiology of hypertension and cardiac failure in 2K1C hypertensive rats.
 
Article
This work was designed in order to gain an insight on the mechanisms by which antioxidants prevent pancreatic disorders. We have examined the properties of cinnamtannin B-1, which belongs to the class of polyphenols, against the effect of hydrogen peroxide (H(2)O(2)) in mouse pancreatic acinar cells. We have studied Ca(2+) mobilization, oxidative state, amylase secretion, and cell viability of cells treated with cinnamtannin B-1 in the presence of various concentrations of H(2)O(2). We found that H(2)O(2) (0.1-100 μM) increased CM-H(2)DCFDA-derived fluorescence, reflecting an increase in oxidation. Cinnamtannin B-1 (10 μM) reduced H(2)O(2)-induced oxidation of CM-H(2)DCFDA. CCK-8 induced oxidation of CM-H(2)DCFDA in a similar way to low micromolar concentrations of H(2)O(2), and cinnamtannin B-1 reduced the oxidant effect of CCK-8. In addition, H(2)O(2) induced a slow and progressive increase in intracellular free Ca(2+) concentration ([Ca(2+)](c)). Cinnamtannin B-1 reduced the effect of H(2)O(2) on [Ca(2+)](c), but only at the lower concentrations of the oxidant. H(2)O(2) inhibited amylase secretion in response to cholecystokinin, and cinnamtannin B-1 reduced the inhibitory action of H(2)O(2) on enzyme secretion. Finally, H(2)O(2) reduced cell viability, and the antioxidant protected acinar cells against H(2)O(2). In conclusion, the beneficial effects of cinnamtannin B-1 appear to be mediated by reducing the intracellular Ca(2+) overload and intracellular accumulation of digestive enzymes evoked by ROS, which is a common pathological precursor that mediates pancreatitis. Our results support the beneficial effect of natural antioxidants in the therapy against oxidative stress-derived deleterious effects on cellular physiology.
 
Article
Genetic contribution of tumor necrosis factor polymorphism (TNF-alpha-308G/A) in patients with juvenile idiopathic arthritis (JIA) on response to TNF blocking agents, as well as matrix metalloproteinase-9 (MMP-9) production, is not yet well established. We have investigated whether the TNF-alpha-308G/A polymorphism can influence MMP-9 level and clinical response to etanercept (TNF receptor II-Fc fusion protein) in JIA patients, after 1 year of treatment. A total of 66 patients with polyarticular JIA and 65 healthy children were screened for the polymorphism using the polymerase chain reaction-restriction fragment length polymorphism method. JIA patients donated paired blood samples prior to and 12 months after etanercept therapy. Plasma MMP-9 level was determined using an enzyme-linked immunosorbent assay kit. Clinical assessment was performed according to ACR Pedi 50 improvement criteria. The frequency of the A allele was significantly higher in JIA patients compared to controls (39% vs. 26%, P = 0.026). Patients with the -308GG genotype achieved an ACR Pedi 50 response significantly more frequently than those with the -308AA genotype (P = 0.035). MMP-9 level in patients with the genotype -308GG was significantly decreased after 1 year of treatment with etanercept compared to the value from before (P = 0.036). On the other hand, there was a decrease of MMP-9 levels after treatment, but not statistically significant in patients with the genotypes -308GA/AA. We conclude that etanercept reduces MMP-9 level in children with polyarticular JIA and TNF-alpha-308GG genotype. Our results correlate with findings that the -308A allele is associated with a lower response to etanercept treatment.
 
Adenylylation and phosphorylation of plasma membrane proteins from normal rat liver and AS30D tumor cells. Plasma membranes from normal rat liver (60 µg of protein) (lanes 1 and 3) or AS-30D tumor cells (100 µg of protein) (lanes 2 and 4) were assayed with [α32 P]ATP (lanes 1 and 2) or [γ-32 P]ATP (lanes 3 and 4) and processed by SDS-PAGE and autoradiography. The arrow points to the major adenylylated and phosphorylated 115 kDa protein from AS-30D tumor cells (lanes 2 and 4). To obtain similar labeling intensities, the exposure time of the autoradiographs of the adenylylated proteins in AS-30D tumor cells (lane 2) was 3 times longer than that of their normal rat liver counterparts (lane 1). The exposure times of the phosphorylated proteins from normal rat liver (lane 3) and AS-30D tumor cells (lane 4) were identical. 
Article
We have identified in plasma membrane fractions isolated from rat hepatocarcinoma AS-30D ascites cells three glycoproteins of 125 kDa, 115 kDa and 105 kDa (gp125, gp115 and gp105) which become adenylylated using ATP as substrate, most readily in the presence of EDTA. The gp115 becomes also phosphorylated. The adenylylation of these tumor glycoproteins was much lower than that of a group of analogous adenylylatable glycoproteins (gp130, gp120-gp110 dimer and gp100) present in normal rat liver plasma membrane. The tumor glycoproteins were reversibly O-adenylylated at threonine residues, as was the case for their normal rat liver counterparts. The tumor gp115, and the gp120-gp110 dimer from normal rat liver were both isolated using either ATP-affinity chromatography and/or AMP-affinity chromatography. The gp120-gp110 dimer from normal rat liver was identified as the plasma cell differentiation antigen-1 (PC-1 protein), an ecto-5' phosphodiesterase/ nucleotide-pyrophosphatase (5'-PDE/NPPase). The gp115 from tumor cells also exhibited Zn2+-stimulated 5'-PDE and NPPase activities in alkaline conditions, although it appears to be distinct from the PC-1 protein. We have determined that the gp115 is an ecto-enzyme that catalyzes the hydrolysis of extracellular ATP, since its adenylylation and phosphorylation were detected in intact cells using extracellularly added [alpha-32P]ATP or [gamma-32P]ATP, respectively, in the absence of any permeabilizing agent.
 
Article
Beta3-adrenergic agonists are well-recognited to promote lipid mobilisation and adipose tissue remodeling in rodents, leading to multilocular fat cells enriched in mitochondria. However, effects of beta3-adrenergic agonists on glucose transport are still controversial. In this work, we studied in white adipose tissue (WAT) the influence of sustained beta3-adrenergic stimulation on the glucose transport and on the mitochondrial monoamine oxidase (MAO) activity. As one-week administration of CL 316243 (CL, 1 mg/kg/d) induces beta-adrenergic desensitization in rat but not in guinea pig adipocytes, attention was paid to compare these models. When expressing glucose uptake as nmoles of 2-deoxyglucose/100 mg cell lipids, maximally stimulated uptake was increased in adipocytes of WAT from treated rats but not from treated guinea pigs. However, basal hexose uptake was also increased in CL-treated rats and, as a consequence, the dose-dependent curves for insulin stimulation were similar in control and CL-treated rats when expressed as fold increase over basal. Insulin-induced lipogenesis was unchanged in rat or guinea pig adipocytes after CL-treatment. The glucose carriers GLUT4 and corresponding mRNA were increased in subcutaneous WAT or in brown adipose tissue (BAT) but not in visceral WAT or muscles of CL-treated rats. There was an increase of MAO activity in WAT and BAT, but not in liver, of CL-treated rats while no change was detected in guinea pigs. These findings show that only rat adipocytes, which are beta3-adrenergic-responsive, respond to chronic beta3-AR agonist by an increase of GLUT4 content and MAO activity, despite a desensitization of all beta-adrenoceptor subtypes.
 
Article
Beta3-adrenergic agonists have been considered as potent antiobesity and antidiabetic agents mainly on the basis of their beneficial actions discovered twenty years ago in obese and diabetic rodents. The aim of this work was to verify whether prolonged treatment with a beta3-adrenergic agonist known to stimulate lipid mobilisation, could promote desensitization of beta-adrenergic responses. Wistar rats and guinea pigs were treated during one week with CL 316243 (CL, 1 mg/kg/d) by implanted osmotic minipumps. In control animals, beta3-adrenergic agonists were lipolytic in rat but not in guinea pig adipocytes. CL-treatment did not alter body weight gain in both species, but reduced fat stores in rats. Lipolysis stimulation by forskolin was unmodified but responses to beta1-, beta2- and beta3-agonists were reduced in visceral or subcutaneous white adipose tissues of CL-treated rats. Similarly, the beta3-adrenergic-dependent impairment of insulin action on glucose transport and lipogenesis in rat adipocytes was diminished after CL-treatment. In rat adipocytes, [125I]ICYP binding and beta3-adrenoceptor mRNA levels were reduced after sustained CL administration. These findings show that CL 316243 exerts (beta3-adrenergic lipolytic and antilipogenic effects in rat adipocytes. These actions, which are likely involved in the fat depletion observed in rat, also lead to the desensitization of all beta-adrenergic responses. Therefore this desensitization, together with the lack of slimming action in guinea pig, seriously attenuates the usefulness of beta3-agonists as antiobesity agents, and may explain why such agonists have not been conducted to a widespread clinical use.
 
Article
Biogenic amines like tyramine, methylamine and the non-naturally occuring amine, benzylamine, have been described to promote adipose conversion of murine 3T3 preadipocytes. To further investigate these novel effects of amines, we studied whether they selectively mimic the long-term adipogenic action of insulin. To this aim, we decided to use the 3T3-L1 cell line since this model needs a complex combination of inducers to trigger the differentiation programme: insulin, isobutylmethylxanthine (IBMX, an activator of cAMP-signal transduction pathway) and the synthetic glucocorticoid, dexamethasone. A cell culture protocol was designed, by which each component of the differentiation cocktail was replaced with either benzylamine or tyramine, in order to determine whether these amine oxidase substrates could substitute any of the differentiation inducers in 3T3-L1 cells. The incomplete lipid accumulation found in cells grown under IBMX- or dexamethasone-free conditions was not improved by the daily addition of amines to the culture medium. Insulin was the only component of adipose differentiation cocktail of 3T3-L1 that could be replaced, although partially, by tyramine or benzylamine. When used at 0.5 mM, these amines resulted in a significant increase of triacylglycerol accumulated eight days after confluence, when compared to cells kept without insulin. This partial insulin replacement was totally abolished by SSAO-inhibitors, while MAO-blockade did not reduce lipid accumulation. As previously reported for other insulin-sensitive processes, such as stimulation of glucose transport or lipolysis inhibition in mature adipocytes, the stimulation of adipogenesis by tyramine and benzylamine was an SSAO-dependent mechanism that apparently shared common signaling pathways with insulin.
 
Article
Lipoic acid (LA) is a naturally occurring compound with antioxidant properties. Recent attention has been focused on the potential beneficial effects of LA on obesity and related metabolic disorders. Dietary supplementation with LA prevents insulin resistance and upregulates adiponectin, an insulin-sensitizing adipokine, in obese rodents. The aim of this study was to investigate the direct effects of LA on adiponectin production in cultured adipocytes, as well as the potential signaling pathways involved. For this purpose, fully differentiated 3T3-L1 adipocytes were treated with LA (1-500 μM) during 24 h. The amount of adiponectin secreted to media was detected by ELISA, while adiponectin mRNA expression was determined by RT-PCR. Treatment with LA induced a dose-dependent inhibition on adiponectin gene expression and protein secretion. Pretreatment with the PI3K inhibitor LY294002 inhibited adiponectin secretion and mRNA levels, and significantly potentiated the inhibitory effect of LA on adiponectin secretion. The AMPK activator AICAR also reduced adiponectin production, but surprisingly, it was able to reverse the LA-induced inhibition of adiponectin. The JNK inhibitor SP600125 and the MAPK inhibitor PD98059 did not modify the inhibitory effect of LA on adiponectin. In conclusion, our results revealed that LA reduces adiponectin secretion in 3T3-L1 adipocytes, which contrasts with the stimulation of adiponectin described after in vivo supplementation with LA, suggesting that an indirect mechanism or some in vivo metabolic processing is involved.
 
Article
Plasma level of the protein VAP-1/SSAO (Vascular Adhesion Protein-1/Semicarbazide-Sensitive Amine Oxidase) is increased in diabetes and/or obesity and may be related to vascular complications associated to these pathologies. The aim of this work was to complete a preceding study where we described the role played by some hormones or metabolites, implicated in diabetes and/or obesity, in the regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes. Here we focused on the previously observed effect produced by TNFalpha in the release of VAP-1/SSAO and studied the effect of a beta-adrenergic compound, isoproterenol. Both compounds stimulated the release of VAP-1/SSAO to the culture medium but had a different effect on the VAP-1/SSAO membrane form. While TNFalpha produced a decrease on VAP-1/SSAO membrane form content, isoproterenol did not modify it. We thus observed two different ways of regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes by metabolites implicated in diabetes and adipose tissue physiopathology. Our work permits a better understanding of this increased plasma VAP-1/SSAO levels observed in diabetes.
 
Article
Lipoic acid (LA) is an antioxidant with therapeutic properties on several diseases like diabetes and obesity. Apelin is a novel adipokine with potential beneficial actions on glucose metabolism and insulin resistance. The aim of this study was to examine in 3T3-L1 adipocytes the effects of LA on apelin gene expression and secretion, as well as elucidate the signaling pathways involved. We also tested the regulation of adipose apelin gene expression by LA supplementation in a model of high-fat diet-induced obesity. LA increased apelin secretion but not apelin gene expression in 3T3-L1 adipocytes. The AMPK inhibitor Compound C induced an increase in LA-stimulated apelin production, and, on the contrary, the AMPK activator AICAR completely reversed the LA stimulatory effects on apelin secretion, also inducing a significant reduction in apelin mRNA levels in this in vitro model. Apelin mRNA levels were increased in those animals fed with the high-fat diet, while the caloric restriction decreased apelin mRNA to control levels. However, apelin gene expression was not significantly modified in rats treated with LA compared with the obese group. The current data suggest the ability of LA to modulate apelin secretion by adipocytes. However the insulin-sensitizing effect of LA in vivo is not related to changes in apelin gene expression in our model of diet-induced obesity.
 
Mitochondrial network gene and protein expression. Mitochondrial biogenesis and function-related (a) and networkrelated (b) gene expression. Total RNA was extracted from 3T3-L1 culture dishes at days 0, 3, 6, 9, and 12 (n=5 to 6). Samples were analyzed by quantitative RT-PCR, and all PCR 
3T3-L1 cellular oxygen consumption during adipose differentiation process (nmol O 2 /min/mg total protein): (a) basal cellular respiration in non-permeabilized cells; (b) oligomycin cellular respiration; (c) FCCP cellular respiration; (d) respiratory control ratio (RCR). DiOC6 fluorescence (membrane potential marker) during 3T3-L1 adipogenesis in basal (e) and oligomycin-treated cells (f). *p<0.05 from day 0, excluding day 3, n=6 
Measurements in whole cell culture dishes at each step of differentiation. a ROS quantification, (b) NO quantification, (c) citrate synthase activity, (d) COX activity. *p<0.05 from day 0, n=6 
Article
Mitochondria have been shown to be impaired in insulin resistance-related diseases but have not been extensively studied during the first steps of adipose cell development. This study was designed to determine the sequence of changes of the mitochondrial network and function during the first days of adipogenesis. 3T3-L1 preadipocytes were differentiated into adipocytes without using glitazone compounds. At days 0, 3, 6, 9, and 12, mitochondrial network imaging, mitochondrial oxygen consumption, membrane potential, and oxidative phosphorylation efficiency were assessed in permeabilized cells. Gene and protein expressions related to fatty acid metabolism and mitochondrial network were also determined. Compared to preadipocytes (day 0), new adipocytes (days 6 and 9) displayed profound changes of their mitochondrial network that underwent fragmentation and redistribution around lipid droplets. Drp1 and mitofusin 2 displayed a progressive increase in their gene expression and protein content during the first 9 days of differentiation. In parallel with the mitochondrial network redistribution, mitochondria switched to uncoupled respiration with a tendency towards decreased membrane potential, with no variation of mtTFA and NRF1 gene expression. The expression of PGC1α and NRF2 genes and genes involved in lipid oxidation (UCP2, CD36, and CPT1) was increased. Reactive oxygen species (ROS) production displayed a nadir at day 6 with a concomitant increase in antioxidant enzyme gene expression. This 3T3-L1-based in vitro model of adipogenesis showed that mitochondria adapted to the increased number of lipid droplets by network redistribution and uncoupling respiration. The timing and regulation of lipid oxidation-associated ROS production appeared to play an important role in these changes.
 
Intracellular and extracellular ROS content in control and hyperoxia treatments at 24 h in 3T3-L1 adipocytes (14 days postdifferentiation). White bar control group, gray bar hyperoxia
Effect of 95 % oxygen exposure on glucose uptake, glycerol, and lactate release in 3T3-L1 adipocytes (14 days postdifferentiation). White bars control group, gray bars hyperoxia group. Data ( n 0 6) are expressed as mean±SD. U Mann – Whitney test was performed to identify statistical effects. * P <0.05 and ** P <0.001 vs untreated cells 
Gene expression analysis of HIF-1 α , leptin, IL-6, MCP- 1, PPAR- γ , adiponectin, and ANGPTL4 at 24 h of 95 % oxygen treatment in 3T3-L1 adipocytes (14 days postdifferentiation). White bars control group, gray bars hyperoxia group (HPx). A 
Article
Adipose tissue often becomes poorly oxygenated in obese subjects. This feature may provide cellular mechanisms involving chronic inflammation processes such as the release of pro-inflammatory cytokines and macrophage infiltration. In this context, the purpose of the present study was to determine whether a hyperoxia exposure on mature adipocytes may influence the expression of some adipokines and involve favorable changes in specific metabolic variables. Thus, 3T3-L1 adipocytes (14 days differentiated) were treated with 95 % oxygen for 24 h. Cell viability, intra and extracellular reactive oxygen species (ROS) content, glucose uptake, as well as lactate and glycerol concentrations were measured in the culture media. Also, mRNA levels of hypoxia-inducible factor (HIF)-1α, leptin, interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, peroxisome proliferator-activated receptor (PPAR)-γ, adiponectin, and angiopoietin-related protein (ANGPTL)4 were analyzed. Hyperoxia treatment increased intra and extracellular ROS content, reduced glucose uptake and lactate release and increased glycerol release. Additionally, a higher oxygen tension led to an upregulation of the expression of IL-6, MCP-1, and PPAR-γ, while ANGPTL4 was downregulated in the hyperoxia group with respect to control. The present data shows that hyperoxia treatment seems to produce an inflammatory response due to the release of ROS and the upregulation of pro-inflammatory adipokines, such as IL-6 and MCP-1. On the other hand, hyperoxia may have an indirect effect on insulin sensitivity due to the upregulation of PPAR-γ signaling as well as a possible modulation of both glucose and lipid metabolic markers. To our knowledge, this is the first study analyzing the effect of hyperoxia in 3T3-L1 adipocytes.
 
Article
Since during pregnancy the mother switches from an anabolic to a catabolic condition, the present study was addressed to determine the effect of 48 h food deprivation on days 7, 14 and 20 of pregnancy in the rat as compared to age matched virgin controls. Body weight, free of conceptus, decreased with food deprivation more in pregnant than in virgin rats, with fetal weight (day 20) also diminishing with maternal starvation. The decline of plasma glucose with food deprivation was greatest in 20 day pregnant rats. Insulin was highest in fed 14 day pregnant rats, and declined with food deprivation in all the groups, the effect being not significant in 7-day pregnant rats. Food deprivation increased plasma glycerol only in virgin and 20 day pregnant rats. Plasma NEFA and 3-hydroxybutyrate increased with food deprivation in all groups, the effect being highest in 20 day pregnant rats. Food deprivation decreased plasma triacylglycerols in 14 day pregnant rats but increased in 20 day pregnant rats. In 20-day fetuses, plasma levels of glucose, NEFA and triacylglycerols were lower than in their mothers when fed, and food deprivation caused a further decline in plasma glucose, whereas both NEFA and 3-hydroxybutyrate increased. Liver triacylglycerols concentration did not differ among the groups when fed, whereas food deprivation caused an increase in all pregnant rats and fetuses, the effect being highest in 20-day pregnant rats. Lipoprotein lipase (LPL) activity in adipose tissue was lower in 20 day pregnant rats than in any of the other groups when fed, and it decreased in all the groups with food deprivation, whereas in liver it was very low in all groups when fed and increased with food deprivation only in 20 day pregnant rats. A significant increase in liver LPL was found with food deprivation in 20 day fetuses, reaching higher values than their mothers. Thus, the response to food deprivation varies with the time of pregnancy, being lowest at mid pregnancy and greatest at late pregnancy, and although fetuses respond in the same direction as their mothers, they show a specific response in liver LPL activity.
 
Article
The rising incidence of prostate cancer and benign prostatic hypertrophy in the Western world is a cause of increasing public health concern. The most active androgen in the prostate is 5α-dihydrotestosterone obtained from testosterone (T) by the enzyme 5α-reductase (5α-R), expressed in the prostate as two isozymes, 5α-R1 and 5α-R2. These isozymes are involved in the growth and development of normal prostate and in the onset and progression of prostate disease. Besides androgens, prolactin (PRL) may also play a role, although it is not clear whether its effects on the prostate are in synergism with or independent of those of androgens. We previously demonstrated that sulpiride, an inductor of hyperprolactinemia, increased mRNA levels of 5α-R isozymes in prostate of adult rat. We hypothesized a possible interrelationship between PRL levels and 5α-R, although the effects of sulpiride per se cannot be ruled out. In the present study, one-step quantitative reverse transcription polymerase chain reaction coupled with laser-induced fluorescence capillary electrophoresis was used to quantify mRNA levels of both 5α-R isozymes in prostate of adult rat after administration of metoclopramide (MTC), another inductor of PRL secretion. With the administration regimens studied, MTC produced an increase in prostate weight and mRNA levels of 5α-R1 and 5α-R2 in adult rats. Given our finding that MTC per se or MTC-induced hyperprolactinemia modifies prostate disease-related parameters in animals with reduced plasma T levels, further investigation is warranted into the possibility that MTC use by aging males may increase their risk of prostate disease.
 
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Creatine is an ergogenic aid used in individual and team sports. The aim of this study is to analyze the effect of monohydrate creatine supplementation on physical performance during 6 consecutive maximal speed 60 meter races, and the changes induced in some characteristic biochemical and ventilatory parameters. The study was carried out on nineteen healthy and physically active male volunteers, and randomly distributed into two groups: Group C received a supplement of creatine monohydrate (20 g/day for 5 days) and group P received placebo. Tests were performed before and after supplementation. No significant changes were observed in weight or body water measured by bioimpedance or the sum of 7 skinfold or performance during the 60 meter races. Group C showed a statistically significant increase in plasma creatinine from 69.8 +/- 12.4 to 89.3 +/- 12.4 micromol x L(-1) (p<0.05). In group C in the second control day (after creatine supplementation), expiratory volume V(E), O2 uptake and CO2 production were lower after 2 minutes of active recovery period. These results indicate that creatine monohydrate supplementation does not appear to improve the performance in 6 consecutive 60 meter repeated races but may modify ventilatory dynamics during the recovery after maximal effort.
 
Article
The use of microarrays to evaluate the transcriptome has transformed our view of biology. In addition to the focused, hypothesis-testing studies that we have traditionally conducted in cell biology, we are now able to see global changes within the entire system of the cell in response to a treatment. By examining a biological question under multiple complementary perturbations model systems (e.g. yeast, C. Elegans) have revealed new complexity that would have been impossible to see on a gene-by-gene approach. Unfortunately, beyond the use of transcript profiles to define the molecular signature of diseases (e.g. cancer), transcriptomics has not been extensively used to study intestinal biology. This review will provide a roadmap for effective use of gene expression profiling for biological research and will review some of the microarray work that has been done to better understand the nature of intestinal development and enterocyte differentiation.
 
Article
High-fat diet (HFD) elevates circulatory fatty acids and influences glucose and fat metabolism. Azelaic acid (AzA), a naturally occurring α,ω-dicarboxylic acid in wheat, rye, barley, oat seeds and sorghum, has been reported to exert antidiabetic effects in HFD-induced type 2 diabetes mellitus (T2DM) C57BL/6J mice. The present study was undertaken to identify the genes that are differentially modulated by treatment with AzA in HFD-fed mice. Mice were fed HFD for 10 weeks and subjected to intragastric administration of 80 mg/kg body weight (BW) of AzA daily along with HFD from 11 to 15 weeks. Lipid profile, adipokines and cytokines were examined in the plasma/liver of mice. Whole genome profiling was performed in the liver of mice using microarray and validated by qRT-PCR, Western blot and immunohistochemical analyses. HFD intake resulted in significantly elevated lipids (except high-density lipoproteins), resistin, tumour necrosis factor alpha and interleukin-6 with marked reduction in adiponectin. Administration of AzA to HFD-fed mice significantly restored the lipids, adipokines and cytokines to near normal. Transcript profiling revealed that HFD intake activated the genes involved in stress response, cell cycle regulation and apoptosis. Treatment with AzA caused increased expression of genes involved in reactive oxygen species (ROS) scavenging, receptor-mediated signalling, transcription, protein modification and insulin signal transduction. AzA activates insulin signal molecules leading to insulin sensitivity. The ability of AzA to modulate the expression of these genes supports the notion that AzA is a promising drug candidate for the treatment of insulin resistance associated with T2DM.
 
Article
In this study, we have investigated the effect of the nutritive phytochemicals, indole-3-carbinol (I3C) and its metabolite, 3, 3'- diindolylmethane (DIM) on oxidative stress developed in type 2 diabetes mellitus (T2DM). This work was carried out in the genetically modified mouse (C57BL/6J mice) that closely simulated the metabolic abnormalities of the human disease after the administration of high fat diet (HFD). Glucose, insulin, hemoglobin (Hb), glycated hemoglobin (HbA1c), thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH), conjugated dienes (CD), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), vitamin C, vitamin E, and reduced glutathione (GSH) levels were monitored in all the groups. Treatments positively modulate the glucose, insulin, and Hb and HbA1c levels in HFD mice. TBARS, LOOH, and CD were decreased in treatment groups when compared to the HFD group. Treatments increase SOD, CAT, GPx levels (erythrocyte, liver, kidney, and heart) and vitamin C, vitamin E, and GSH levels (plasma, liver, kidney, and heart) in diabetic mice. From the study, it was clear that the antioxidant-scavenging action were accelerated in mice treated with DIM than the I3C treatment group which was comparable with the standard drug metformin.
 
Article
Heat shock protein (HSP) 70 plays a critical role in protecting the heart from various stressor-induced cell injuries; the mechanism remains to be further understood. The present study aims to elucidate the effect of a probiotics-derived protein, LGG-derived protein p75 (LGP), in alleviating the ischemia/reperfusion (I/R)-induced heart injury. We treated rats with the I/R with or without preadministration with LGP. The levels of HSP70 and carboxy terminus of HSP70-interacting protein (CHIP) in the heart tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of CHIP on suppression of HSP70 and the effect of LGP on suppression of CHIP were investigated with an I/R rat model and a cell culture model. The results showed that I/R-induced infarction in the heart could be alleviated by pretreatment with LGP. HSP70 was detected in naïve rat heart tissue extracts. I/R treatment significantly suppressed the level of HSP70 and increased the levels of CHIP in the heart. A complex of CHIP/HSP70 was detected in heart tissue extracts. The addition of recombinant CHIP to culture inhibited HSP70 in heart cells. LGP was bound CHIP in heart cells and prevented the CHIP from binding HSP70. In summary, I/R can suppress HSP70 and increase CHIP in heart cells. CHIP can suppress HSP70 that can be prevented by pretreatment with LGP. The results imply that CHIP may be a potential target in the prevention of I/R-induced heart cell injury.
 
Article
The present work was aimed at studying the effects of a subchronic lithium treatment on rat liver and kidneys, paying attention to the relationship between lithium toxicity, oxidative stress, and stress protein expression. Male rats were submitted to lithium treatment by adding 2 g of lithium carbonate/kg of food for different durations up to 1 month. This treatment led to serum concentrations ranging from 0.5 mM (day 7) to 1.34 mM (day 28) and renal insufficiency highlighted by an increase of blood creatinine and urea levels and a decrease of urea excretion. Lithium treatment was found to trigger an oxidative stress both in kidney and liver, leading to an increase of lipid peroxidation level (TBARS) and of superoxide dismutase and catalase activities. Conversely, glutathione peroxidase activity was reduced. Constitutive HSP73 (heat shock protein 73) expression was not modified by lithium treatment, whereas inducible HSP72 was down-regulated in kidney. GRP94 (glucose regulated protein 94) appeared as two isoforms of 92 and 98 kDa: the 98-kDa protein being overexpressed in kidney by lithium treatment whereas 92-kDa protein was underexpressed both in kidney and liver.
 
Differences on triacylglycerols (A) and lipoprotein (a) (B) plasma concentration (mg/dl) according to body fat mass tertiles and the-174G/C polymorphism of the IL6 gene. *p<0.05; **p<0.01.
Article
It has been shown that NADPH oxidase plays a role in oxidative stress which has been involved in the development of metabolic syndrome. The -930A/G polymorphism of the CYBA gene (that codes p22phox, a major component of the NADPH oxidase) has been associated with human hypertension and with a reduction in NADPH oxidase activity. In this work, we have examined the influence of the -930A/G polymorphism on obesity risk and insulin resistance in a case-control study of Spanish subjects (n=313). In the obese group (n=159), there was a statistically significant association between the GG genotype of the -930A/G polymorphism of the CYBA gene and fasting insulin levels and HOMA index. This outcome agrees with previous findings concerning functional analyses of this polymorphism and reinforces the hypothesis that insulin resistance is associated with oxidative stress. In conclusion, a protective effect in carriers of the -930A/G polymorphism of the p22phox gene against insulin resistance in a population of Spanish obese adults has been found.
 
Top-cited authors
Martinez Alfredo
  • Universidad de Navarra
Quan Zou
  • Xiamen University
Yong Huang
Xing-Jia Shen
  • Jiangsu University of Science and Technology
Maria Moreno-Aliaga
  • Universidad de Navarra