RESUMO
Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the outcome of such screening, this study compared the effect of 60 plant extracts on the activity of PPO from mushroom ( Agaricus bisporus , AbPPO) and PPO from potato ( Solanum tuberosum , StPPO). Some plant extracts had different effects on the two PPOs: an extract that inhibited one PPO could be an activator for the other. As an example of this, the mate ( Ilex paraguariensis ) extract was investigated in more detail. In the presence of mate extract, oxygen consumption by AbPPO was found to be reduced >5-fold compared to a control reaction, whereas that of StPPO was increased >9-fold. RP-UHPLC-MS analysis showed that the mate extract contained a mixture of phenolic compounds and saponins. Upon incubation of mate extract with StPPO, phenolic compounds disappeared completely and saponins remained. Flash chromatography was used to separate saponins and phenolic compounds. It was found that the phenolic fraction was mainly responsible for inhibition of AbPPO and activation of StPPO. Activation of StPPO was probably caused by activation of latent StPPO by chlorogenic acid quinones.
Assuntos
Agaricus/enzimologia , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/metabolismo , Inibidores Enzimáticos/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Ilex paraguariensis/química , Extratos Vegetais/química , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/metabolismo , Solanum tuberosum/enzimologia , Fenóis/química , Saponinas/químicaRESUMO
Sulfite salts are widely used as antibrowning agents in food processing. Nevertheless, the exact mechanism by which sulfite prevents enzymatic browning has remained unknown. Here, we show that sodium hydrogen sulfite (NaHSO3) irreversibly blocks the active site of tyrosinase from the edible mushroom Agaricus bisporus, and that the competitive inhibitors tropolone and kojic acid protect the enzyme from NaHSO3 inactivation. LC-MS analysis of pepsin digests of NaHSO3 -treated tyrosinase revealed two peptides showing a neutral loss corresponding to the mass of SO3 upon MS(2) fragmentation. These peptides were found to be homologous peptides containing two of the three histidine residues that form the copper-B-binding site of mushroom tyrosinase isoform PPO3 and mushroom tyrosinase isoform PPO4, which were both present in the tyrosinase preparation used. Peptides showing this neutral loss behavior were not found in the untreated control. Comparison of the effects of NaHSO3 on apo-tyrosinase and holo-tyrosinase indicated that inactivation is facilitated by the active site copper ions. These data provide compelling evidence that inactivation of mushroom tyrosinase by NaHSO3 occurs through covalent modification of a single amino-acid residue, probably via addition of HSO3(-) to one of the copper-coordinating histidines in the copper-B site of the enzyme.
Assuntos
Agaricus/enzimologia , Cobre/metabolismo , Histidina/metabolismo , Monofenol Mono-Oxigenase/antagonistas & inibidores , Sulfitos/farmacologia , Agaricus/efeitos dos fármacos , Sequência de Aminoácidos , Antioxidantes/farmacologia , Sítios de Ligação , Ligação Competitiva , Domínio Catalítico , Cromatografia Líquida , Cinética , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Micotoxinas/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Pironas/farmacologia , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Tropolona/farmacologiaRESUMO
The antibrowning activity of sodium hydrogen sulfite (NaHSO(3)) was compared to that of other sulfur-containing compounds. Inhibition of enzymatic browning was investigated using a model browning system consisting of mushroom tyrosinase and chlorogenic acid (5-CQA). Development of brown color (spectral analysis), oxygen consumption, and reaction product formation (RP-UHPLC-PDA-MS) were monitored in time. It was found that the compounds showing antibrowning activity either prevented browning by forming colorless addition products with o-quinones of 5-CQA (NaHSO(3), cysteine, and glutathione) or inhibiting the enzymatic activity of tyrosinase (NaHSO(3) and dithiothreitol). NaHSO(3) was different from the other sulfur-containing compounds investigated, because it showed a dual inhibitory effect on browning. Initial browning was prevented by trapping the o-quinones formed in colorless addition products (sulfochlorogenic acid), while at the same time, tyrosinase activity was inhibited in a time-dependent way, as shown by pre-incubation experiments of tyrosinase with NaHSO(3). Furthermore, it was demonstrated that sulfochlorogenic and cysteinylchlorogenic acids were not inhibitors of mushroom tyrosinase.
Assuntos
Agaricales/enzimologia , Ácido Clorogênico/química , Inibidores Enzimáticos/química , Aditivos Alimentares/química , Proteínas Fúngicas/química , Monofenol Mono-Oxigenase/química , Compostos de Enxofre/química , Oxirredução , Sulfitos/químicaRESUMO
The effect of sodium hydrogen sulfite (S), used as antibrowning agent, on the phenolic profile of potato extracts was investigated. This extract was compared to one obtained in the presence of ascorbic acid (A). In the presence of A, two major compounds were obtained, 5-O-caffeoylquinic acid (5-CQA) and 4-O-caffeoyl quinic acid. With S, their 2'-sulfo-adducts were found instead, the structures of which were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Also, for minor caffeoyl derivatives and quercetin glycosides, the corresponding sulfo-adducts were observed. Feruloyl and sinapoyl derivatives were not chemically affected by the presence of S. Polyphenol oxidase (PPO) was thought to be responsible for the formation of the sulfo-adducts. This was confirmed by preparing 2'-sulfo-5-O-caffeoyl quinic acid in a model system using 5-CQA, sodium hydrogen sulfite, and PPO. This sulfo-adduct exhibited a small bathochromic shift (λmax 329 nm) as compared to 5-CQA (λmax 325 nm) and a strong hypochromic shift with an extinction coefficient of 9357±395 M(-1) cm(-1) as compared to 18494±196 M(-1) cm(-1), respectively. The results suggest that whenever S is used as an antibrowning agent, the O-quinone formed with PPO reacts with S to produce sulfo-O-diphenol, which does not participate in browning reactions.
Assuntos
Reação de Maillard/efeitos dos fármacos , Fenóis/metabolismo , Extratos Vegetais/metabolismo , Solanum tuberosum/metabolismo , Sulfitos/farmacologia , Compostos de Enxofre/metabolismo , Catecol Oxidase/metabolismo , Ácido Clorogênico/análogos & derivados , Ácido Clorogênico/metabolismo , Ácido Quínico/análogos & derivados , Ácido Quínico/metabolismo , Solanum tuberosum/efeitos dos fármacosRESUMO
Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with a liquid chromatography/mass spectrometry (LC/MS)-based screening method. The glyceollins I-III and glyceollidins I-II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the (1,3) A(+) retro-Diels-Alder (RDA) fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the five pterocarpans in negative-ion (NI) mode were proposed, which involved the cleavage of the C-ring and/or D-ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H(2) O(x,y) RDA fragments, whereas its presence gave predominantly the common RDA fragments.
Assuntos
Cumarínicos/química , Extratos Vegetais/química , Pterocarpanos/química , Rhizopus/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Oxilipinas/química , Prenilação , Plântula/química , Plântula/microbiologia , Glycine max/química , Glycine max/microbiologiaRESUMO
Human IGF-I was over-expressed in skeletal muscles of C57/BL6xCBA mice under the control of the rat skeletal alpha-actin gene promoter. RT-PCR verified expression of the transgene in skeletal muscle but not in the liver of 1- and 21-day old heterozygote transgenic mice. The concentration of endogenous mouse IGF-I, measured by an immunoassay which does not detect human IGF-I, was not significantly different between transgenic mice and wild-type littermates (9.5 +/- 0.8 and 13.3 +/- 1.9 ng/g in muscle; 158.3 +/- 18.6 and 132.9 +/- 33.1 ng/ml in plasma, respectively). In contrast, quantitation with antibodies to human IGF-I showed an increase in IGF-I of about 100 ng/ml in plasma and 150 ng/g in muscle of transgenic mice at 6 months of age. Transgenic males, compared to their age matched wild-type littermates, had a significantly higher body weight (38.6 +/- 0.53 g vs. 35.8 +/- 0.64 g at 6 months of age; P < 0.001), dry fat-free carcass mass (5.51 +/- 0.085 vs. 5.08 +/- 0.092 g; P < 0.001) and myofibrillar protein mass (1.62 +/- 0.045 vs. 1.49 +/- 0.048 g; P < 0.05), although the fractional content of fat in the carcass was lower (167 +/- 7.0 vs. 197 +/- 7.7 g/kg wet weight) in transgenic animals. There was no evidence of muscle hypertrophy and no change in the proportion of slow type I myofibres in the limb muscles of Rskalpha-actin/hIGF-I transgenic mice at 3 or 6 months of age. Phenotypic changes in Rskalpha-actin/hIGF-I mice are likely to be due to systemic as well as autocrine/paracrine effects of overproduction of IGF-I due to expression of the human IGF-I transgene. The effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was tested on: (i) muscle regeneration in auto-transplanted whole muscle grafts; (ii) myofibre atrophy following sciatic nerve transection; and (iii) sarolemmal damage and myofibre necrosis in dystrophic mdx muscle. No beneficial effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was seen in these three experimental models.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Músculo Esquelético/fisiologia , Distrofias Musculares/genética , Regeneração/genética , Actinas/genética , Animais , Peso Corporal , Feminino , Humanos , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like I/genética , Masculino , Camundongos , Camundongos Transgênicos , Denervação Muscular , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Distrofias Musculares/patologia , Regiões Promotoras Genéticas , Ratos , Ativação TranscricionalRESUMO
1. Protein utilisation and turnover were measured in male chickens sampled from a line selected for high breast yield and a randombred control line (lines QL and CL, experiment 1) and in male chickens sampled from lines selected for either high or low abdominal fatness (lines FL and LL, experiment 2). In each experiment, 18 birds per line were given iso-energetic (12.9 MJME/kg) diets containing either 120 or 220 g CP/kg from 21 to 29 d (experiment 1) and 33 to 43 d (experiment 2). 2. Measurements were made of growth rate, food intake, body composition, excreta production and Ntau-methylhistidine excretion as a measure of myofibrillar protein breakdown, and fractional rates (%/d) of protein deposition, breakdown and synthesis were calculated. 3. In experiment 1, there were no significant differences between the line means for the fractional measures of protein turnover, but there was marked differential response in the two lines in the fractional rates of protein deposition, breakdown and synthesis, to increase in protein intake. The positive slope of the regressions of fractional (%/d) protein deposition and synthesis rates on protein intake (g/d/kg BW) were approximately 1.4- and 2.0-fold higher respectively in the QL than the CL line birds, and the negative slope of the regression of fractional breakdown rate on protein intake was approximately threefold greater in the CL than the QL line birds. 4. In experiment 2, fractional deposition rate was 6.2% lower, but fractional breakdown rate 9.4% higher in the LL than the FL birds, whilst there was essentially no difference in response of the FL and LL birds in the components of protein turnover to increase in protein intake. Line differences in deposition and breakdown rates were thus a reflection of the considerably higher (20%) food and hence protein intake in the FL than the LL birds. 5. The differential line responses in protein turnover in the two experiments suggest that selection for increased breast muscle yield and for reduced body fatness manipulate different physiological pathways in relation to protein turnover, but neither selection strategy results in an improvement in net protein utilisation at typical levels of protein intake by birds on commercial broiler diets, through a reduction in protein breakdown rate.
Assuntos
Composição Corporal/genética , Galinhas/genética , Proteínas Alimentares/metabolismo , Tecido Adiposo/fisiologia , Ração Animal , Animais , Composição Corporal/fisiologia , Galinhas/fisiologia , Dieta , Ingestão de Alimentos , Masculino , Músculo Esquelético/fisiologia , Aumento de PesoRESUMO
The recovery of radioactivity in the urine of guineapigs following a bolus intravenous dose of chromatographically pure 14C-Ntau-methylhistidine was measured in order to test whether the excretion of Ntau-methylhistidine (Ntau-MH) is a valid index of myofibrillar protein breakdown in these animals. Four male and four female guineapigs were dosed and after 7 days, 91.65+/-2.82% and 3.58+/-0.91% of injected radioactivity was recovered in the excreta and tissues, respectively. The average total recovery of 95.2+/-3.0% was not significantly different from 100%. Male guineapigs excreted the radioactivity more slowly than females (70% of the dose excreted within 74 h vs 39 h, respectively) but cumulative excretion at 7 days was the same for each sex. Chromatographic analysis of the urine showed almost all of the radioactivity to be associated with a single peak corresponding to Ntau-MH, indicating a lack of significant metabolism. These data show that although the clearance of 14C-Ntau-MH is slower than in rats or humans the urinary excretion of Ntau-MH is a valid index for myofibrillar protein degradation in the guineapig.
Assuntos
Cobaias/urina , Metilistidinas/urina , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Actinas/metabolismo , Animais , Radioisótopos de Carbono , Feminino , Masculino , Taxa de Depuração Metabólica , Metilistidinas/farmacocinética , Miosinas/metabolismo , Caracteres SexuaisRESUMO
Insulin-like growth factor-I (IGF-I) is known to have anabolic effects in freely fed rats. We have investigated the ability of infused LR(3)IGF-I, an analogue of IGF-I, to attenuate the loss of lean tissue due to food restriction in young (5 weeks) and adult (12 weeks) rats. Groups of rats received food at 100%, 78%, 56% or 33% of ad libitum levels. Within each nutrition group the rats were continuously infused with LR(3)IGF-I at (98 nmol/day)/kg body weight or vehicle for 7 days. At each level of food intake, rats infused with LR(3)IGF-I maintained higher body weight (around 3-8%;P< 0.001) and nitrogen retention (P< 0.001) than those infused with vehicle alone but muscle protein was not conserved. LR(3)IGF-I infusion increased fat loss only in young rats (P< 0.05) despite a reduction in plasma insulin levels in both age groups (P< 0.01). Muscle protein turnover rates were unaffected by LR(3)IGF-I in young rats. In adult rats LR(3)IGF-I exacerbated the effects of food restriction through increased rates of protein breakdown, reduced RNA content and reduced rates of protein synthesis (P< 0.05) despite their larger fat reserves. Although young and adult rats show differing metabolic responses, we conclude that infusion of LR(3)IGF-I to either group during short-term food restriction does not ameliorate the loss of lean tissue by allowing more efficient utilization and/or partitioning of nutrients.
Assuntos
Peso Corporal/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Músculo Esquelético/efeitos dos fármacos , Fatores Etários , Animais , Composição Corporal/efeitos dos fármacos , Alimentos , Insulina/sangue , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Músculo Esquelético/anatomia & histologia , Tamanho do Órgão/efeitos dos fármacos , Condicionamento Físico Animal , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Ratos , Ratos WistarRESUMO
The efficacy of exogenous IGFs to stimulate growth and modulate protein and fat deposition was examined in a number of broiler chicken lines. From around 600 g body weight the chickens received a continuous infusion of vehicle (0.1 M acetic acid), human recombinant IGF-I or [Gly1]IGF-II at 300 microg/kg body weight per day, or a combined infusion of 150 microg/kg/day of each IGF for 2 weeks. Experiment 1 used commercial broiler female chickens and included measurements of nitrogen balance, Ntau-methylhistidine excretion and muscle protein synthesis rates. In Experiment 2 the same treatments were applied to three experimental lines of chickens selected for high food consumption (relatively fat), high food utilisation efficiency (relatively lean), or at random (control). IGF-I, but not IGF-II, significantly increased growth rate and food utilisation efficiency by around 10-15% in each experiment, an effect which was consistent across all genotypes. Nitrogen balance was significantly increased by IGF-I in Experiment 1 as was carcass nitrogen content in Experiment 2, indicating that the increased growth was in lean tissue. Carcass fat was consistently reduced in chickens receiving IGF-I and was related to the levels of circulating IGF-I (r2 = 0.30, P < 0.01) but not triiodothyronine. Protein synthesis rates were unaffected by treatment and could not account for increased growth rate. However, there was a significant reduction in Ntau-methylhistidine excretion indicating a reduced rate of muscle protein breakdown in IGF-I-treated chickens (1. 56%/day vs 2.05%/day for IGF-I-treated vs controls, P < 0.05). The efficiency of feed utilisation was inversely related to the rate of protein breakdown (r2 = 0.25, P < 0.01). In conclusion, these experiments are the first to report an enhancement of growth and food utilisation efficiency by broiler chickens receiving exogenous IGF-I. Our results show that IGF-I may be important in controlling the growth and efficiency of food utilisation of young chickens at least in part by modulating the rates of protein breakdown.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Galinhas/crescimento & desenvolvimento , Fator de Crescimento Insulin-Like II/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Animais , Glicemia/efeitos dos fármacos , Composição Corporal/efeitos dos fármacos , Galinhas/sangue , Feminino , Humanos , Bombas de Infusão Implantáveis/veterinária , Insulina/sangue , Fator de Crescimento Insulin-Like I/administração & dosagem , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/administração & dosagem , Fator de Crescimento Insulin-Like II/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Tri-Iodotironina/sangue , Tri-Iodotironina/efeitos dos fármacosRESUMO
The anti-catabolic efficacy of IGF-I treatment commencing before, with or after the onset of catabolism was compared in order to test whether earlier treatment can more effectively blunt a catabolic response. Young rapidly growing male rats (145 g body weight) and old weight-stable female rats (270 g body weight) were used in two experiments. The IGF-I variant LR 3IGF-I was continuously infused at 280 micrograms/day from 2 days before (early), concurrent with or 2 days after (delayed) commencement of a 6 day dexamethasone infusion (125 micrograms/kg per day). Both early and delayed treatment of young rats with LR 3IGF-I significantly reduced the measured catabolic effects of dexamethasone. Early treatment was more effective than delayed treatment, with significantly higher nitrogen balance (90 +/- 12 vs 31 +/- 6 mg/day), carcass nitrogen gain (0.37 +/- 0.27 vs -1.13 +/- 0.10% per day) and protein fractional synthesis rates after 2 (10.4 +/- 0.5 vs 8.3 +/- 0.2% per day) and 6 days of dexamethasone (8.2 +/- 0.6 vs 7.0 +/- 0.3% per day). Similarly, fractional breakdown rates of carcass protein were maintained at significantly lower levels in rats given early treatment (6.23 +/- 0.24 vs 6.60 +/- 0.22% per day). In contrast, the old rats were less responsive to LR 3IGF-I treatment and only early and concurrent treatment significantly reduced the catabolic response, partly because of higher food intake. Early treatment was superior to delayed treatment and led to significantly higher nitrogen balance(-19 +/- 11 vs -83 +/- 7 mg/day) and carcass nitrogen fractional gain (-1.19 +/- 0.40 vs -2.76 +/- 0.37% per day) as well as lower final rates of carcass protein fractional breakdown (3.55 +/- 0.15 vs 3.83 +/- 0.07% per day). These experiments show that early (prophylactic) treatment with IGF-I is superior to delayed treatment for reducing catabolism induced by dexamethasone. The results provide the basis for further research to determine if prophylactic IGF-I treatment is useful in other circumstances where catabolism can be anticipated, such as in elective major surgery.
Assuntos
Dexametasona/farmacologia , Substâncias de Crescimento/uso terapêutico , Crescimento/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/análogos & derivados , Envelhecimento/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Fator de Crescimento Insulin-Like I/uso terapêutico , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Nitrogênio/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Proteínas Recombinantes/uso terapêutico , Análise de Regressão , Fatores de TempoRESUMO
The relative acute hypoglycaemic potencies of IGF-I and several variants of IGF-I which bind poorly to the IGF-I binding proteins (IGFBPs) have been examined in marmosets (Callithrix jacchus) and the pig. In the marmoset study, IGF-I and des(1-3)IGF-I were compared in anaesthetised and conscious animals in a range of bolus doses from 42 to 270 micrograms/kg body weight. In the pig study, IGF-I was compared with four variants, des(1-3)IGF-I long-IGF-I, R3IGF-I and long-R3IGF-I (LR3IGF-I), which show reduced affinity for the IGFBPs as well as with insulin. Doses in the pig were 20 and 50 micrograms/kg body weight for the IGFs and 3 micrograms/kg for insulin. In each study serial blood samples were taken from 30 min before to 4 h after the bolus injection. Plasma glucose levels were decreased in a dose-responsive manner with the pig more sensitive than either the conscious or anaesthetised marmoset (maximum lowering 4.8, 3.7 and 2.5 mmol/l respectively). The IGF variants were consistently 2- to 3-fold more potent than IGF-I in each animal for lowering of plasma glucose to the nadir, with the potency reflecting the relative affinities for binding to the IGFBPs and the IGF-I receptors. Thus, hypoglycaemic potency was in the order IGF-I < long-IGF-I < R3IGF-I approximately LR3IGF-I < des (1-3)IGF-I. Notably the variants suppressed plasma glucose levels over a much longer period than did IGF-I, the cumulative suppression over four hours showing an approximately 4- to 8-fold increase in the extent of hypoglycaemia. The prolonged suppression was not simply proportional to the hypoglycaemic nadir; at doses equipotent for glucose lowering, the cumulative hypoglycaemic effect for the variants in either species was about 2-fold that for IGF-I. The differential effect of the variants in the marmoset could not be accounted for by correlated changes in plasma insulin, IGF-I or IGFBP levels in plasma. Indirect effects via inhibition of glucagon, or direct effects via hepatic insulin receptors are postulated to account for the results. There was a dose-related reduction in plasma amino acids in the pig but, unlike the case for plasma glucose, only one analogue, LR3IGF-I was more potent than IGF-I. The response to LR3IGF-I was accentuated at the high dosage but on the basis of the other variants tested this effect could not be ascribed to either of the incorporated molecular variations. Despite their more rapid clearance from the circulation, variants of IGF-I which show lower affinity for binding to IGFBPs show proportionately superior potency for sustained hypoglycaemic action. Since our data were obtained in animal models of accepted relevance to humans these results point to the possible superior efficacy of the variants, especially des(1-3)IGF-I, over IGF-I for use as an adjunct to insulin treatment of hyperglycaemic conditions.
Assuntos
Glicemia/análise , Hipoglicemiantes/farmacologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Aminoácidos/sangue , Análise de Variância , Animais , Área Sob a Curva , Callithrix , Relação Dose-Resposta a Droga , Hipoglicemiantes/metabolismo , Insulina/sangue , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Proteínas Recombinantes/farmacologia , Suínos , Fatores de TempoRESUMO
Des(1-3)IGF-I, a truncated variant of human IGF-I with the tripeptide Gly-Pro-Glu absent from the N-terminus, has been isolated from bovine colostrum, human brain and porcine uterus. This protein probably results from post-translational cleavage of IGF-I. Des(1-3)IGF-I generally is about 10-fold more potent than IGF-I at stimulating hypertrophy and proliferation of cultured cells, a consequence of much reduced binding to IGF-binding proteins, in turn caused by the absence of the glutamate at position 3. The increased potency is retained in part when the variant is administered in vivo, with selective anabolic effects particularly evident in gut tissues. Clinical opportunities for des(1-3)IGF-I have not yet been evaluated, but could apply in catabolic states as well as for the treatment of inflammatory bowel diseases.
Assuntos
Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Fator de Crescimento Insulin-Like I/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Processamento de Proteína Pós-Traducional , SuínosRESUMO
The relative potency of IGF-I and the analogue LR3IGF-I to either promote growth or reverse catabolism in rats when administered by injection rather than by continuous infusion has been examined. LR3IGF-I has very low affinity for the IGF-binding proteins in the rat and hence is cleared from the circulation more quickly than is IGF-I. Experiments were performed in normal growing rats (150 g body weight) and in rats made catabolic by dexamethasone infusion (20 micrograms/day). IGFs or vehicle were delivered subcutaneously for 7 days either by continuous infusion via osmotic pumps or by injection once or twice daily at 320 and 400 micrograms/day in normal and catabolic rats respectively. As expected, continuous infusion of IGFs showed greater efficacy than either of the injection modes especially in its anti-catabolic actions. When infused continuously LR3IGF-I was generally 1.5- to 2-fold more potent than IGF-I for changes in body weight gain, visceral organ weights and feed use efficiency. Notably, LR3IGF-I remained more potent than IGF-I in several of these effects even when the peptides were given by once-daily injection. In addition, N tau-methylhistidine excretion by dexamethasone-treated rats was reduced to a threefold greater extent by injected LR3IGF-I than by injected IGF-I. Notwithstanding these effects, LR3IGF-I was barely equipotent with IGF-I for reversal of carcass muscle loss in dexamethasone-treated rats. Despite its more rapid clearance from the circulation, injected LR3IGF-I retains superior potency to injected IGF-I for several actions, albeit the potency is much reduced compared with continuous infusion. Thus our data indicate that use of IGF analogues which have low affinity for binding proteins may have advantages in potency and/or tissue specificity where IGFs are necessarily administered by injection.
Assuntos
Crescimento/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/administração & dosagem , Animais , Peso Corporal/efeitos dos fármacos , Dexametasona , Bombas de Infusão Implantáveis , Injeções Subcutâneas , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/farmacologia , Intestino Delgado/anatomia & histologia , Masculino , Músculo Esquelético/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Baço/anatomia & histologia , Timo/anatomia & histologiaRESUMO
While numerous researchers have used rat models to investigate the in vivo actions of IGF-I, interpretation of the results in terms of true concentrations of rat IGF-I (rIGF-I) in plasma has been hampered by the absence of homologous reference standards. In order to overcome this we have produced recombinant rIGF-I (rrIGF-I) from Escherichia coli using procedures similar to those we have previously described for the production of other recombinant IGFs. The rrIGF-I is indistinguishable from serum-derived rIGF-I when characterized in a number of in vitro assays including ability to stimulate protein synthesis and inhibit protein degradation in cultured rat cells, as well as in interactions with the rat type-1 IGF receptor and with rat IGF-binding proteins. Moreover, both the serum-derived and the recombinant rat proteins are similar to recombinant human IGF-I (rhIGF-I) in these assays. However, differences between the human and rat IGFs are apparent when tested in immunoassays using some antibodies raised against rhIGF-I. Furthermore, the differences between rhIGF-I and rrIGF-I are even greater when rhIGF-I is used as the competing radiolabel in these assays, a situation that can lead to a two- to threefold underestimation of the actual concentration of IGF-I in rat plasma. These results indicate that, while immunoassays employing antibodies raised against rhIGF-I and rhIGF-I reference standards reliably indicate trends in IGF-I concentrations in rat plasma, the true amounts of rIGF-I present can only be assured in an assay using homologous tracer and reference peptides.
Assuntos
Fator de Crescimento Insulin-Like I/química , Animais , Sequência de Bases , Imunoensaio , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/isolamento & purificação , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligonucleotídeos/genética , Ligação Proteica , Engenharia de Proteínas , Ratos , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
We have examined the influence of nutrition on plasma IGF-I, IGF-II and IGF-binding protein (IGFBP) levels and on hepatic IGF-I gene expression in young meat-type chickens. Plasma IGF concentrations were measured by using RIA with recombinant chicken IGFs as standards. In chickens fed the control diet containing 200 g/kg dietary protein ad libitum for 7 days, plasma IGF-I concentrations increased significantly from those found in the initial control group. Food restriction for either 4 or 7 days decreased plasma IGF-I by 30% from the initial control. When chickens were refed ad libitum for 3 days after 4 days of restricted feeding, plasma IGF-I levels recovered to those of the control birds fed ad libitum. In chickens eating a low protein diet (100 g/kg protein), the plasma IGF-I tended to be lowered but the decrease was not significant. Although the intensity of IGF-I and beta-actin mRNA bands protected in the RNase protection assay was changed by nutrition, no statistical effect of nutrition on the ratio of IGF-I to beta-actin was observed. The nutritional treatments had no effect on plasma IGF-II concentrations. Western ligand blot and chromatographic analyses were used to investigate the influence of nutrition on IGFBP profiles. Both IGF-I and IGF-II ligands in the Western ligand blot revealed the most intense binding at 30 kDa for plasma obtained from chickens with restricted food intake. The 30 kDa band also appeared at a lower intensity in the group fed a low protein diet but not in any other groups. These observations were confirmed by neutral gel chromatography. The chicken IGF-II ligand revealed an intensely labelled band corresponding to 75 kDa and this was not affected by nutrition. IGF-I and IGFBP concentrations in the plasma of young broiler chickens were influenced by nutritional state but IGF-II concentrations were not. The lack of a response in circulating IGF-II levels may have been due to the presence of high concentrations of a 75 kDa specific binding protein which did not respond to nutrition in this experiment.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Galinhas/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/sangue , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/metabolismo , Animais , Western Blotting , Galinhas/sangue , Jejum/sangue , Feminino , Fator de Crescimento Insulin-Like I/genética , RNA Mensageiro/metabolismo , RadioimunoensaioRESUMO
Studies in diabetic rodents and humans provide evidence that IGF-I may alleviate the diabetic state and insulin resistance to some degree. To assess the efficacy of IGFs as an adjunct treatment with insulin in diabetes, we infused IGF-I or des(1-3)IGF-I for 7 days at 0, 10.7, 26.7, and 66.8 nmol/day to streptozotocin-induced diabetic rats in conjunction with infusions of 0, 2.2, 5.6, or 14 nmol/day insulin. Both insulin and des(1-3)IGF-I increased body weight gain by 7 g/day compared with controls (1.2 g/day), but there was no additive effect. However, for nitrogen retention, the effects of des(1-3)IGF-I were additive with those of 2.2 nmol/day insulin. Des(1-3)IGF-I was two- to threefold more potent than IGF-I. At comparable rates of total nitrogen retention, carcass nitrogen retention was approximately 35% higher with insulin than with IGF treatment, indicating a differential tissue response. IGFs did not alter carcass fat content. Des(1-3)IGF-I increased liver glycogen additively with insulin but reduced glucosuria only when given with 5.6 nmol insulin per day, indicating the possibility of a facilitatory effect, perhaps via increased insulin sensitivity. Insulin was 10- to 25-fold more potent in these glucoregulatory actions. Differential effects of the hormones were also observed for kidney, liver, and thymus weights. We conclude that IGFs and especially the more potent des(1-3)IGF-I may have a role as an adjunct to insulin therapy in diabetic patients.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Fator de Crescimento Insulin-Like I/administração & dosagem , Insulina/administração & dosagem , Animais , Glicemia/metabolismo , Peso Corporal , Metabolismo dos Carboidratos , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Crescimento/efeitos dos fármacos , Insulina/sangue , Fator de Crescimento Insulin-Like I/metabolismo , Ilhotas Pancreáticas/metabolismo , Metabolismo dos Lipídeos , Proteínas Musculares/metabolismo , Nitrogênio/metabolismo , Tamanho do Órgão , Ratos , Ratos WistarRESUMO
We have tested whether an animal with substantial amounts of both IGF-I and IGF-II in circulation, such as the guinea pig, would respond to chronic IGF infusion in the same manner as the adult rat, which has negligible amounts of IGF-II in blood. Female guinea pigs of 350 g body weight were continuously infused for 7 days with recombinant guinea pig IGF-I or -II (120 or 360 micrograms/day) or long R3 IGF-I (LR3IGF-I) (120 micrograms/day), an analogue which has much reduced affinities for IGF binding proteins. IGF-I or IGF-II infusion led to substantial increases in plasma IGF-I or IGF-II respectively in comparison with vehicle-infused animals. Nevertheless, body weight gain, feed intake, feed conversion efficiency and carcass composition were not significantly affected by any treatment (significance was deemed to be P < 0.05). Amongst the tissues examined only the fractional weight (g/kg body weight) of the adrenals was increased, and that only by the higher dose (360 micrograms/day) of IGF-I. However, the fractional weight of adrenals, gut, kidneys and spleen were significantly increased by LR3IGF-I, but again overall growth was not stimulated. A possible explanation for the lack of IGF-I effects is that total circulating IGF concentrations were not increased by these treatments. IGF-II significantly raised total IGF concentrations at the higher dose only. Plasma IGF-I was reduced by IGF-II infusion, as was plasma IGF-II by IGF-I infusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/análogos & derivados , Tamanho do Órgão/efeitos dos fármacos , Somatomedinas/metabolismo , Glândulas Suprarrenais/anatomia & histologia , Animais , Feminino , Cobaias , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like II/farmacologia , Intestinos/anatomia & histologia , Rim/anatomia & histologia , Baço/anatomia & histologiaRESUMO
OBJECTIVE: To compare different aspects of intermediary metabolism in self perceived 'small-eating' females and self-perceived near normal weight 'large-eating' females and relate the data to those reported for Pima Indians who have the world's highest prevalence of non-insulin dependent diabetes mellitus and obesity. DESIGN: Make repeat measurements of rates of oxygen consumption, carbon dioxide production and blood metabolites in 'large-' and 'small-eating' females at rest, during different activities and after ingestion of a standardised liquid meal. SUBJECTS: Nine self perceived, 'large-eating' females and nine self perceived 'small-eating' females. MEASUREMENTS: Resting metabolic rates (RMR), respiratory quotient (RQ) values and plasma insulin, glucagon insulin-like growth factor (IGF-1), dehydroepiandrosterone sulphate (DHEA-SO4) and glucose. RESULTS: RMR (adjusted for FFM) averaged 3891 +/- 93 J/min in the 'small-eaters' and 3375 +/- 107 J/min in the 'large-eaters' for ten consecutive measurements conducted at 30 min intervals during the control period for the measurement of the thermic effect of food. Over this period the average RQ for the 'small-eating' women (0.81) was significantly greater than that of the 'large-eating' women (0.78). The two groups responded similarly to an oral glucose tolerance test but the concentration of DHEA-SO4 in plasma was 35% higher in the 'small-eaters'. CONCLUSION: The 'small-eating' women may have a greater risk of weight gain but they counteract this tendency by maintaining high activity levels.