A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea)

Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata) genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/µg of protein) of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit.

The alga Bryothamnion seaforthii contains carbohydrates with antinociceptive activity

Bryothamnion seaforthii, a red alga common to the Northeastern coast of Brazil, was used to prepare the protein fraction F0/60 by ammonium sulfate precipitation. The chromatography of F0/60 on DEAE-Sephadel column resulted in two lectin fractions, PI and PII, which have antinociceptive properties in rodents. We determined the antinociceptive activity of the PII fraction and of a carbohydrate-containing fraction (CF) in mice. The CF was prepared from the dried algae, after digestion with 100 mM sodium acetate, pH 6.0, containing 5 mM cysteine, EDTA and 0.4% papain, at 60ºC. A 10% cetylpyridinium chloride was added to the filtrate, and the precipitate was dissolved with 2 M NaCl:ethanol (100:15, v/v) followed by the carbohydrate precipitation with ethanol. The final precipitate, in acetone, was dried at 25ºC. The PII fraction markedly inhibited acetic acid-induced abdominal writhing after ip administration (control: 27.1 ± 2.20; PII 0.1 mg/kg: 5.5 ± 1.85; 1 mg/kg: 1.6 ± 0.72 writhes/20 min) and after oral administration (control: 32.0 ± 3.32; PII 0.1 mg/kg: 13.1 ± 2.50; 1 mg/kg: 9.4 ± 3.96 writhes/20 min). PII was also effective against both phases of pain induced by 1% formalin (control, ip: 48.2 ± 2.40 and 27.7 ± 2.56 s; PII: 1 mg/kg, ip: 34.3 ± 5.13 and 5.6 ± 2.14 s; control, po: 44.5 ± 3.52 and 25.6 ± 2.39 s; PII 5 mg/kg, po: 26.5 ± 4.67 and 15.3 ± 3.54 s for the 1st and 2nd phases, respectively) and in the hot-plate test. The CF (ip) also displayed significant antinociceptive properties in all tests but at higher doses (1 and 5 mg/kg, ip and po). Thus, CF at the dose of 5 mg/kg significantly inhibited writhes (ip: 7.1 ± 2.47 and po: 14.5 ± 2.40 writhes/20 min) as well as the 1st (po: 19.6 ± 1.74 s) and 2nd (po: 7.1 ± 2.24 s) phases of the formalin test compared to controls ip and po. The antinociceptive effects of both the PII and CF in the formalin and hot-plate tests were prevented at least partially by pretreatment with the opioid receptor antagonist naloxone (2 mg/kg, sc). Moreover, both fractions retained antinociceptive activity in the acetic acid-induced writhing test following heating, a procedure which abolished the hemagglutinating activity of the fraction, presumably due to lectins also present. Finally, both fractions also prolonged the barbiturate-induced sleeping time. These results indicate that carbohydrate molecules present in the PII (26.8% carbohydrate) and CF (21% of the alga dried weight) obtained from B. seaforthii display pronounced antinociceptive activity which is resistant to heat denaturation and is mediated by an opioid mechanism, as indicated by naloxone inhibition.

Differential effect of plant lectins on mast cells of different origins

Histamine release induced by plant lectins was studied with emphasis on the carbohydrate specificity, external calcium requirement, metal binding sites, and mast cell heterogeneity and on the importance of antibodies bound to the mast cell membrane to the lectin effect. Peritoneal mast cells were obtained by direct lavage of the rat peritoneal cavity and guinea pig intestine and hamster cheek pouch mast cells were obtained by dispersion with collagenase type IA. Histamine release was induced with concanavalin A (Con A), lectins from Canavalia brasiliensis, mannose-specific Cymbosema roseum, Maackia amurensis, Parkia platycephala, Triticum vulgaris (WGA), and demetallized Con A and C. brasiliensis, using 1-300 µg/ml lectin concentrations applied to Wistar rat peritoneal mast cells, peaking on 26.9, 21.0, 29.1, 24.9, 17.2, 10.7, 19.9, and 41.5%, respectively. This effect was inhibited in the absence of extracellular calcium. The lectins were also active on hamster cheek pouch mast cells (except demetallized Con A) and on Rowett nude rat (animal free of immunoglobulins) peritoneal mast cells (except for mannose-specific C. roseum, P. platycephala and WGA). No effect was observed in guinea pig intestine mast cells. Glucose-saturated Con A and C. brasiliensis also released histamine from Wistar rat peritoneal mast cells. These results suggest that histamine release induced by lectins is influenced by the heterogeneity of mast cells and depends on extracellular calcium. The results also suggest that this histamine release might occur by alternative mechanisms, because the usual mechanism of lectins is related to their binding properties to metals from which depend the binding to sugars, which would be their sites to bind to immunoglobulins. In the present study, we show that the histamine release by lectins was also induced by demetallized lectins and by sugar-saturated lectins (which would avoid their binding to other sugars). Additionally, the lectins also released histamine from Rowett nude mast cells that are free of immunoglobulins.

How does yeast respond to pressure?

The brewing and baking yeast Saccharomyces cerevisiae has been used as a model for stress response studies of eukaryotic cells. In this review we focus on the effect of high hydrostatic pressure (HHP) on S. cerevisiae. HHP exerts a broad effect on yeast cells characteristic of common stresses, mainly associated with protein alteration and lipid bilayer phase transition. Like most stresses, pressure induces cell cycle arrest. Below 50 MPa (500 atm) yeast cell morphology is unaffected whereas above 220 MPa wild-type cells are killed. S. cerevisiae cells can acquire barotolerance if they are pretreated with a sublethal stress due to temperature, ethanol, hydrogen peroxide, or pressure. Nevertheless, pressure only leads to protection against severe stress if, after pressure pretreatment, the cells are also re-incubated at room pressure. We attribute this effect to the inhibition of the protein synthesis apparatus under HHP. The global genome expression analysis of S. cerevisiae cells submitted to HHP revealed a stress response profile. The majority of the up-regulated genes are involved in stress defense and carbohydrate metabolism while most repressed genes belong to the cell cycle progression and protein synthesis categories. However, the signaling pathway involved in the pressure response is still to be elucidated. Nitric oxide, a signaling molecule involved in the regulation of a large number of cellular functions, confers baroprotection. Furthermore, S. cerevisiae cells in the early exponential phase submitted to 50-MPa pressure show induction of the expression level of the nitric oxide synthase inducible isoform. As pressure becomes an important biotechnological tool, studies concerning this kind of stress in microorganisms are imperative.

Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15% SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.

Opposite lipemic response of Wistar rats and C57BL/6 mice to dietary glucose or fructose supplementation

The metabolic effects of carbohydrate supplementation in mice have not been extensively studied. In rats, glucose- and fructose-rich diets induce hypertriacylglycerolemia. In the present study, we compared the metabolic responses to two monosaccharide supplementations in two murine models. Adult male Wistar rats (N = 80) and C57BL/6 mice (N = 60), after 3 weeks on a standardized diet, were submitted to dietary supplementation by gavage with glucose (G) or fructose (F) solutions (500 g/L), 8 g/kg body weight for 21 days. Glycemia was significantly higher in rats after fructose treatment (F: 7.9 vs 9.3 mM) and in mice (G: 6.5 vs 10 and F: 6.6 vs 8.9 mM) after both carbohydrate treatments. Triacylglycerolemia increased significantly 1.5 times in rats after G or F supplementation. Total cholesterol did not change with G treatment in rats, but did decrease after F supplementation (1.5 vs 1.4 mM, P < 0.05). Both supplementations in rats induced insulin resistance, as suggested by the higher Homeostasis Model Assessment Index. In contrast, mice showed significant decreases in triacylglycerol (G: 1.8 vs 1.4 and F: 1.9 vs 1.4 mM, P < 0.01) and total cholesterol levels (G and F: 2.7 vs 2.5 mM, P < 0.05) after both monosaccharide supplementations. Wistar rats and C57BL/6 mice, although belonging to the same family (Muridae), presented opposite responses to glucose and fructose supplementation regarding serum triacylglycerol, free fatty acids, and insulin levels after monosaccharide treatment. Thus, while Wistar rats developed features of plurimetabolic syndrome, C57BL/6 mice presented changes in serum biochemical profile considered to be healthier for the cardiovascular system.

Beneficial effects of exercise training (treadmill) on insulin resistance and nonalcoholic fatty liver disease in high-fat fed C57BL/6 mice

C57BL/6 mice develop signs and symptoms comparable, in part, to the human metabolic syndrome. The objective of the present study was to evaluate the effects of exercise training on carbohydrate metabolism, lipid profile, visceral adiposity, pancreatic islet alterations, and nonalcoholic fatty liver disease in C57BL/6 mice. Animals were fed one of two diets during an 8-week period: standard (SC, N = 12) or very high-fat (HF, N = 24) chow. An exercise training protocol (treadmill) was then established and mice were divided into SC and HF sedentary (SC-Sed, HF-Sed), exercised groups (SC-Ex, HF-Ex), or switched from HF to SC (HF/SC-Sed and HF/SC-Ex). HF/HF-Sed mice had the greatest body mass (65% more than SC/SC-Sed; P < 0.0001), and exercise reduced it by 23% (P < 0.0001). Hepatic enzymes ALP (+80%), ALT (+100%) and AST (+70%) were higher in HF/HF mice than in matched SC/SC. Plasma insulin was higher in both the HF/HF-Sed and HF/SC-Sed groups than in the matched exercised groups (+85%; P < 0.001). Pancreatic islets, adipocytes and liver structure were greatly affected by HF, ultimately resulting in islet β-cell hypertrophy and severe liver steatosis. The HF group had larger islets than the SC/SC group (+220%; P < 0.0001), and exercise significantly reduced liver steatosis and islet size in HF. Exercise attenuated all the changes due to HF, and the effects were more pronounced in exercised mice switched from an HF to an SC diet. Exercise improved the lipid profile by reducing body weight gain, visceral adiposity, insulin resistance, islet alterations, and fatty liver, contributing to obesity and steatohepatitis control.

Effects of chronic treadmill training on body mass gain and visceral fat accumulation in overfed rats

This study evaluated the effects of chronic treadmill training on body mass gain and visceral fat accumulation in overfed rats. Overfeeding was induced by reducing the litter size to 3 male pups per mother during the suckling period. The litter size of control rats was adjusted to 10 male pups per mother. Seven weeks after birth overfed and normally fed rats were selected and assigned to a sedentary protocol or to a low-intensity treadmill training protocol (60 min, 5 times/week, for 9 weeks). Four groups (overfed sedentary, N = 23; normally fed sedentary, N = 32; overfed exercised, N = 18, and normally fed exercised, N = 18) were evaluated at 18 weeks. Data are reported as means ± SEM. Initial body weight was similar in control and overfed rats [8.0 ± 0.2 g (N = 42) vs 8.0 ± 0.1 g (N = 50); P > 0.05] and body weight gain during the suckling period was higher in the overfed rats (30.6 ± 0.9 vs 23.1 ± 0.3 g; P < 0.05). Exercise attenuated the body weight gain of overfed compared to sedentary rats (505 ± 14 vs 537 ± 12 g; P < 0.05). The sedentary overfed rats showed higher visceral fat weight compared to normally fed animals (31.22 ± 2.08 vs 21.94 ± 1.76 g; P < 0.05). Exercise reduced visceral fat by 36.5% in normally fed rats and by 35.7% in overfed rats. Exercise attenuated obesity in overfed rats and induced an important reduction of visceral fat.

Time motion analysis of football (soccer) referees during official matches in relation to the type of fluid consumed

The aim of the present study was to determine the effect of volume and composition of fluid replacement on the physical performance of male football referees. Ten referees were evaluated during three official matches. In one match the participants were asked to consume mineral water ad libitum, and in the others they consumed a pre-determined volume of mineral water or a carbohydrate electrolyte solution (6.4% carbohydrate and 22 mM Na+) equivalent to 1% of their baseline body mass (half before the match and half during the interval). Total water loss, sweat rate and match physiological performance were measured. When rehydrated ad libitum (pre-match and at half time) participants lost 1.97 ± 0.18% of their pre-match body mass (2.14 ± 0.19 L). This parameter was significantly reduced when they consumed a pre-determined volume of fluid. Sweat rate was significantly reduced when the referees ingested a pre-determined volume of a carbohydrate electrolyte solution, 0.72 ± 0.12 vs 1.16 ± 0.11 L/h ad libitum. The high percentage (74.1%) of movements at low speed (walking, jogging) observed when they ingested fluid ad libitum was significantly reduced to 71% with mineral water and to 69.9% with carbohydrate solution. An increase in percent movement expended in backward running was observed when they consumed a pre-determined volume of carbohydrate solution, 7.7 ± 0.5 vs 5.5 ± 0.5% ad libitum. The improved hydration status achieved with the carbohydrate electrolyte solution reduced the length of time spent in activities at low-speed movements and increased the time spent in activities demanding high-energy expenditure.

Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

Plasma levels and placental expression of vaspin in pregnant women with diabetes mellitus

The present study aimed to investigate visceral adipose tissue-specific serpin (vaspin) concentrations in serum and term placentas and relate these values to insulin resistance and lipid parameters in women with gestational diabetes mellitus (GDM). A total of 30 GDM subjects and 27 age-matched pregnant women with normal glucose tolerance (NGT, control) were included. Serum glucose, glycated hemoglobin (HbA1c), lipid profile, insulin, and vaspin were measured at the end of pregnancy, and homeostasis model of assessment-insulin resistance (HOMA-IR) values were calculated. Vaspin mRNA and protein levels in placentas were measured by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Serum vaspin levels were significantly lower in the GDM group than in controls (0.49±0.24 vs 0.83±0.27 ng/mL, respectively; P<0.01). Three days after delivery, serum vaspin levels were significantly decreased in subjects with GDM (0.36±0.13 vs0.49±0.24 ng/mL, P<0.01). However, in the GDM group, serum vaspin levels were not correlated with the parameters evaluated. In contrast, in the control group, serum vaspin levels were positively correlated with triglycerides (TG; r=0.45, P=0.02) and very low-density lipoprotein cholesterol (VLDL-C; r=0.42, P=0.03). Placental mRNA vaspin (0.60±0.32 vs0.68±0.32, P=0.46) and protein (0.30±0.08 vs0.39±0.26; P=0.33) levels in the GDM group did not differ significantly from those in the control group, but were negatively correlated with neonatal birth weight in the GDM group (r=-0.48, P=0.03; r=-0.88; P<0.01). Our findings indicated that vaspin may be an important adipokine involved in carbohydrate and lipid metabolism and may also play a role in fetal development.

Cell wall polysaccharides of common beans (Phaseolus vulgaris L.)

The soluble and insoluble cotyledon (SPF-Co and IPF-Co) and tegument (SPF-Te and IPF-Te) cell wall polymer fractions of common beans (Phaseolus vulgaris) were isolated using a chemical-enzymatic method. The sugar composition showed that SPF-Co was constituted of 38.6% arabinose, 23.4% uronic acids, 12.7% galactose, 11.2% xylose, 6.4% mannose and 6.1% glucose, probably derived from slightly branched and weakly bound polymers. The IPF-Co was fractionated with chelating agent (CDTA) and with increasing concentrations of NaOH. The bulk of the cell wall polymers (29.4%) were extracted with 4.0M NaOH and this fraction contained mainly arabinose (55.0%), uronic acid (18.9%), glucose (10.7%), xylose (10.3%) and galactose (3.4%). About 8.7% and 10.6% of the polymers were solubilised with CDTA and 0.01M NaOH respectively and were constituted of arabinose (52.0 and 45.9%), uronic acids (25.8 and 29.8%), xylose (9.6 and 10.2%), galactose (6.1 and 3.9%) and glucose (6.5 and 3.8%). The cell wall polymers were also constituted of small amounts (5.6 and 7.2%) of cellulose (CEL) and of non-extractable cell wall polymers (NECW). About 16.8% and 17.2% of the polymers were solubilised with 0.5 and 1.0M NaOH and contained, respectively, 92.1 and 90.7% of glucose derived from starch (IST). The neutral sugar and polymers solubilization profiles showed that weakly bound pectins are present mainly in SPF-Co (water-soluble), CDTA and 0.01-0.1M NaOH soluble fractions. Less soluble, highly cross-linked pectins were solubilised with 4.0M NaOH. This pectin is arabinose-rich, probably highly branched and has a higher molecular weight than the pectin present in SPF-Co, CDTA and 0.01-0.1M NaOH fractions.

Rheological and structural evaluations of commercial italian salad dressings

The emulsion stability, composition, structure and rheology of four different commercial italian salad dressings manufactured with traditional and light formulations were evaluated. According to the results, the fat content ranged from 8% (w/w) (light) to 34% (w/w) (traditional), the carbohydrate concentration varied between 3.8% (w/w) (traditional) and 14.4% (w/w) (light) and the pH was between 3.6-3.9 for all samples. The microscopic and stability analyses showed that the only stable salad dressing was a light sample, which had the smallest droplet size when compared with the other samples. With respect to the rheological behaviour, all the salad dressings were characterized as thixotropic and shear thinning fluids. However, the stable dressing showed an overshoot at relatively low shear rates. This distinct rheological behavior being explained by the differences in its composition, particularly the presence of a maltodextrin network.

Use of medicinal plant in the manufacturing of energetic cookies

Many nutrients provide energy and regulate physiological processes linked to exercise. This work aimed at using medicinal plant in the manufacturing of energetic cookie. An evaluation of microbiological and physicochemical stability was made within 180 days of the fabrication of the product. Sensorial analysis was performed in all stages with untrained tasters. The results were statistically tested. The evaluation of ergogenic effect of the product used the maximum volume of oxygen index as a parameter through the cooper track test (12 minutes) with 7 voluntaries. The participants received the consent form. This study was approved by the local ethics committee (N° CAAE - 0009.0.313.000-08). It was observed good stability for physicochemical and microbiological parameters. As for sensorial analysis there was a predominance of scores 6 to 8, characterizing good acceptance. It was verified increase in VO2 max after the intake of the cookies with or without the medicinal plants. However, the product with the medicinal plants presented a bigger value, 35.47 VO2 max mL/(kg.min)-1. The fraction of carbohydrate and the presence of medicinal plants can be considered as nutritional ergogenic substances. The lipid fraction favor the energetic aspect of the product. The proposed product presented energetic and ergogenic effect.