Proanthocyanidins potentiate hypothalamic leptin/STAT3 signalling and Pomc gene expression in rats with diet-induced obesity.

OBJECTIVE: Dietary obesity is usually linked with hypothalamic leptin resistance, in which the primary impact is an interference in the homeostatic control of body weight and appetite. Notably, proanthocyanidins (PACs), which are the most abundant phenolic compounds present in human diet, modulate adiposity and food intake. The aim of this study was to assess whether PACs could re-establish appropriate leptin signalling in both the hypothalamus and peripheral tissues. DESIGN: Male Wistar rats were fed either a standard chow diet (STD group, n=7) or a cafeteria diet (CD) for 13 weeks. The CD-fed rats were treated with either grape-seed PAC extract (GSPE) at 25 mg per kg of body weight per day (CD+GSPE group, n=7) or with the vehicle (CD group, n=7) for the last 21 days of the study period. Specific markers for intracellular leptin signalling, inflammation and endoplasmic reticulum stress in the hypothalamus, liver, mesenteric white adipose tissue and skeletal muscle were analysed using immunoblotting and quantitative PCR. RESULTS: GSPE treatment significantly reduced the food intake but did not reverse the hyperleptinemia and body wt gain assessed. However, the animals treated with GSPE exhibited greater hypothalamic activation of signal transducer and activator of transcription-3, which was associated with a rise in the Pomc mRNA levels compared with the CD group. In addition, this restoration of leptin responsiveness was accompanied by lower local inflammation and increased Sirt1 gene expression. The effects of the GSPE treatment in the peripheral tissues were not as evident as those in the hypothalamus, although the GSPE treatment significantly restored the mRNA levels of Socs3 and Ptp1b in the skeletal muscle. CONCLUSIONS: The use of GSPE reduces hyperphagia and improves the central and peripheral leptin resistance associated with diet-induced obesity. Our results suggest that GSPE could exert these effects partially by increasing Sirt1 expression and preventing hypothalamic inflammation.

Grape seed proanthocyanidin supplementation reduces adipocyte size and increases adipocyte number in obese rats.

OBJECTIVES: White adipose tissue (WAT) expands through hypertrophy (increased adipocyte size) and/or hyperplasia (increased adipocyte number). Hypertrophy has been associated with insulin resistance and dyslipidemia independently of body composition and fat distribution. In contrast, hyperplasia protects against metabolic alterations. Proanthocyanidins, which are the most abundant flavonoids in the human diet, improve metabolic disturbances associated with diet-induced obesity without reducing body weight or adiposity. The aim of this study was to determine whether grape seed proanthocyanidin extract (GSPE) can modulate WAT expandability. Because GSPE also contains gallic acid, we also studied the capacity of gallic acid to remodel WAT. DESIGN: Male Wistar rats were fed a standard chow diet (n=6) or a cafeteria diet (CAF) for 11 weeks. After 8 weeks, the CAF-fed animals were supplemented with 25 mg GSPE/kg body weight (n=6), 7 mg gallic acid/kg body weight (n=6) or the vehicle (n=6) for 3 weeks. Histological analyses were performed in the retroperitoneal (rWAT) and inguinal (iWAT) WAT to determine adipocyte size and number. Specific markers for adipogenesis and WAT functionality were analysed in rWAT using quantitative RT-PCR. RESULTS: GSPE or gallic acid supplementation did not reduce weight gain or reverse and adiposity. However, GSPE reduced adipocyte size significantly in rWAT and moderately in iWAT and tripled the adipocyte number in rWAT. Gallic acid slightly reduced adipocyte size in rWAT and iWAT and doubled the adipocyte number in both WATs. In accordance with this adipogenic activity, Pref-1 and PPARγ tended to be overexpressed in rWAT of rats supplemented with GSPE. Moreover, GSPE supplementation increased Plin1 and Fabp4 expression and restored adiponectin expression completely, indicating a better functionality of visceral WAT. CONCLUSIONS: GSPE supplementation has anti-hypertrophic and hyperplasic activities in rats with established obesity, mainly in visceral WAT inducing a healthier expansion of WAT to match the surplus energy provided by the cafeteria diet.

Detection and characterization of silver nanoparticles and dissolved species of silver in culture medium and cells by AsFlFFF-UV-Vis-ICPMS: application to nanotoxicity tests.

A methodology based on Asymmetric Flow Field-Flow Fractionation (AsFlFFF) coupled with UV-Vis absorption spectrometry and ICP mass spectrometry (ICPMS) has been developed and applied to the study of silver nanoparticles (AgNPs) and dissolved species of silver in culture media and cells used in cytotoxicity tests. The effect of a nano-silver based product (protein stabilized silver nanoparticles ca. 15 nm average diameter) on human hepatoma (HepG2) cell viability has been studied. UV-Vis absorption spectrometry provided information about the nature (organic vs. nanoparticle) of the eluted species, whereas the silver was monitored by ICPMS. A shift towards larger hydrodynamic diameters was observed in the AgNPs after a 24 hour incubation period in the culture medium, which suggests a "protein corona" effect. Silver(I) associated with proteins present in the culture medium has also been detected, as a consequence of the oxidation process experimented by the AgNPs. However, the Ag(I) released into the culture medium did not justify the toxicity levels observed. AgNPs associated with the cultured HepG2 cells were also identified by AsFlFFF, after applying a solubilisation process based on the use of tetramethylammonium hydroxide (TMAH) and Triton X-100. These results have been confirmed by transmission electronic microscopy (TEM) analysis of the fractions collected from the AsFlFFF. The effect of AgNPs on HepG2 cells has been compared to that caused by silver(I) as AgNO3 under the same conditions. The determination of the total content of silver in the cells confirms that a much larger mass of silver as AgNPs with respect to AgNO3 (16 to 1) is needed to observe a similar toxicity.

Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver.

OBJECTIVE: To determine whether proanthocyanidins can protect against dyslipidemia induced by a high-fat diet (HFD) and to address the mechanisms that underlie this hypolipidemic effect. DESIGN AND MEASUREMENTS: Female Wistar rats were fed on a HFD for 13 weeks. They were divided into two groups, one of which was treated with a grape seed proanthocyanidin extract (25 mg kg(-1) of body weight) for 10 days. Plasma and liver lipids were measured by colorimetric and gravimetric analysis. Liver, muscle and adipose tissue were used to study the expression of genes involved in the synthesis and oxidation of fatty acids and lipoprotein homeostasis by real-time RT-PCR. RESULTS: The administration of proanthocyanidins normalized plasma triglyceride and LDL-cholesterol (both parameters significantly increased with the HFD) but tended to decrease hypercholesterolemia and fatty liver. Gene expression analyses revealed that proanthocyanidins repressed both the expression of hepatic key regulators of lipogenesis and very low density lipoprotein (VLDL) assembling such as SREBP1, MTP and DGAT2, all of which were overexpressed by the HFD. CONCLUSION: These findings indicate that natural proanthocyanidins improve dyslipidemia associated with HFDs, mainly by repressing lipogenesis and VLDL assembly in the liver, and support the idea that they are powerful agents for preventing and treating lipid altered metabolic states.

Chronic consumption of dietary proanthocyanidins modulates peripheral clocks in healthy and obese rats.

Circadian rhythm plays an important role in maintaining homeostasis, and its disruption increases the risk of developing metabolic syndrome. Circadian rhythm is maintained by a central clock in the hypothalamus that is entrained by light, but circadian clocks are also present in peripheral tissues. These peripheral clocks are trained by other cues, such as diet. The aim of this study was to determine whether proanthocyanidins, the most abundant polyphenols in the human diet, modulate the expression of clock and clock-controlled genes in the liver, gut and mesenteric white adipose tissue (mWAT) in healthy and obese rats. Grape seed proanthocyanidin extracts (GSPEs) were administered for 21 days at 5, 25 or 50 mg GSPE/kg body weight in healthy rats and 25 mg GSPE/kg body weight in rats with diet-induced obesity. In healthy animals, GSPE administration led to the overexpression of core clock genes in a positive dose-dependent manner. Moreover, the acetylated BMAL1 protein ratio increased with the same pattern in the liver and mWAT. With regards to clock-controlled genes, Per2 was also overexpressed, whereas Rev-erbα and RORα were repressed in a negative dose-dependent manner. Diet-induced obesity always resulted in the overexpression of some core clock and clock-related genes, although the particular gene affected was tissue specific. GSPE administration counteracted disturbances in the clock genes in the liver and gut but was less effective in normalizing the clock gene disruption in WAT. In conclusion, proanthocyanidins have the capacity to modulate peripheral molecular clocks in both healthy and obese states.

Moderate red wine consumption protects the rat against oxidation in vivo.

The effect of the moderate consumption of red wine on the antioxidant system in rat liver, kidney and plasma has been evaluated. Wistar rats were treated in separate groups as follows: control; red wine for 45 days or 6 months; and 13.5% ethanol for 45 days or 6 months. The consumption of alcoholic beverages was free because the rat could always choose between the alcoholic beverage and the water. In liver, red wine ingestion resulted in higher hepatic superoxide dismutase and glutathione peroxidase activities after 45 days of treatment. The data indicate that wine and ethanol ingestion resulted in lower hepatic malondialdehyde and enhanced hepatic catalase activity in both of the periods studied. In kidney, the reduced glutathione/oxidized glutathione ratio was higher after 45 days of wine consumption, and the malondialdehyde was lower after 6 months of wine consumption. In plasma, malondialdehyde was lower after 6 months of both treatments, but plasmatic vitamin E was higher after red wine consumption while it was lower after ethanol consumption for this period of time. The present study shows that the moderate and prolonged consumption of red wine is consistent with higher protection against oxidation in vivo.

Model for voluntary wine and alcohol consumption in rats.

It has been suggested that moderate consumption of ethanol and wine has a protective effect on human health. Animal models used to date for alcohol consumption can not mimic real situations in humans because the consumption is forced and/or excessive. The present study proposes to determine the effects of a voluntary and ad lib consumption model more similar to that of human behavior. Male Wistar rats had free access to either standard diet and water or the same diet plus red wine, sweet wine, or a solution equivalent to red wine (13.5% ethanol) or to sweet wine (20% ethanol + 130 g/L sucrose) for 30 days or 6 months. Daily wine consumption was 15.8 +/- 0.9 and 2.0 +/- 0.2 ml/day for sweet and red wines, respectively. The consumption of each of the alcoholic solutions was similar to that of the wine they were simulating. Drinking wine or ethanol did not affect food and water intakes or growth rate. Plasma metabolites were not substantially affected by consumption of wine or ethanol. Although moderate and high wine consumption did not change the activity of plasma marker enzymes of tissue damage, the consumption of the 2 alcoholic solutions caused a long-term increase in the activity of aspartate aminotransferase. It seems that wine consumption protects the organism from hepatic lesions induced by ethanol alone.

Nonalcoholic components in wine reduce low density lipoprotein cholesterol in normocholesterolemic rats.

Using an experimental model that enables the effects of alcohol to be distinguished from the effects of the nonalcoholic components present in wine, we determined whether wine has effects other than those of alcohol on the metabolism of cholesterol. Male rats were fed a standard diet and had free access to water and either wine or an equivalent alcohol solution for 45 d or 6 mon. Alcohol intake was similar in the two groups of animals. Consumption of the alcohol solution or wine did not influence plasma cholesterol or high density lipoprotein-cholesterol. At 45 d, the consumption both of wine and of alcohol solution reduced low density lipoprotein (LDL)-cholesterol and very low density lipoprotein cholesterol. At 6 mon, only the rats that consumed wine had reduced LDL-cholesterol. After 45 d of consuming alcohol solution, total cholesterol in the aorta was significantly increased mainly as a result of the rise in free cholesterol. In the aorta, the effect of wine consumption was similar to the effect of alcohol solution consumption, although it was less intense. The only clear effect that could be ascribed to the nonalcoholic components in wine was that the LDL-cholesterol was reduced in the long term, although aortic cholesterol was not.

Acute administration of grape seed proanthocyanidin extract modulates energetic metabolism in skeletal muscle and BAT mitochondria.

Proanthocyanidin consumption might reduce the risk of developing several pathologies, such as inflammation, oxidative stress and cardiovascular diseases. The beneficial effects of proanthocyanidins are attributed to their antioxidant properties, although they also can modulate gene expression at the transcriptional level. Little is known about the effect of proanthocyanidins on mitochondrial function and energy metabolism. In this context, the objective of this study was to determine the effect of an acute administration of grape seed proanthocyanidin extract (GSPE) on mitochondrial function and energy metabolism. To examine this effect, male Wistar rats fasted for fourteen hours, and then they were orally administered lard oil containing GSPE or were administered lard oil only. Liver, muscle and brown adipose tissue (BAT) were used to study enzymatic activity and gene expression of proteins related to energetic metabolism. Moreover, the gastrocnemius muscle and BAT mitochondria were used to perform high-resolution respirometry. The results showed that, after 5 h, the GSPE administration significantly lowers plasma triglycerides, free fatty acids, glycerol and urea concentrations. In skeletal muscle, GSPE lowers FATP1 mRNA levels and increases mitochondrial oxygen consumption, using pyruvate as the substrate, suggesting a promotion of glycosidic metabolism. Furthermore, GSPE increased the genetic expression of key genes in energy metabolism such as peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1α), and modulated the enzyme activity of proteins, which are involved in the citric acid cycle and electron transport chain (ETC) in BAT. In conclusion, GSPE affects mainly the skeletal muscle and BAT mitochondria, increasing their oxidative capacity rapidly after acute supplementation.

Procyanidin dimer B1 and trimer C1 impair inflammatory response signalling in human monocytes.

The way specific procyanidins exert their anti-inflammatory effects is not fully understood. This study has investigated the capacity of different procyanidins to modulate lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) production in THP1 human monocytes and their effects on the redox regulated protein kinases activity: IkB kinase beta (IKKb) and the extracellular signal-regulated kinase (ERK). LPS-triggered increase of ROS was prevented by cell pre-incubation with procyanidins. LPS induced ERK1/2 activation through phosphorylation, which was inhibited by all the compounds tested, the most active being epigallocatechin (EG), followed by epigallocatechin gallate (EGCG) and C1. Procyanidins inhibited IKKb activity in vitro. C1 and procyanidin extract (PE) exerted the maximal IKKb inhibition, followed by EGCG and dimer B1. Catechin exerted a slight but significant IKKb inhibition, in contrast to epicatechin, which was ineffective. In conclusion, procyanidins reduce the LPS-induced production of ROS and they exert their anti-inflammatory effects by inhibiting ERK1/2 and IKKb activity.

Isoflavone effect on gene expression profile and biomarkers of inflammation.

The use of high throughput techniques to find differences in gene expression profiles between related samples (transcriptomics) that underlie changes in physiological states can be applied in medicine, drug development and nutrition. Transcriptomics can be used to provide novel biomarkers of a future pathologic state and to study how bioactive food compounds or drugs can modulate them in the early stages. In this study, we examine the expression pattern in order to determine the effect of the pathological-inflammatory state on the RAW 264.7 cell model and to ascertain how isoflavones and their active functional metabolites alleviate the inflammatory burst and the extent of gene modulation due to the presence of polyphenols. Results demonstrated that genistein (20 microM) and equol (10 microM) significantly inhibited the overproduction of NO and PGE(2) induced by LPS plus INF-gamma when a pre-treatment was performed or when administered during activation. Daidzein, however, did not exert similar effects. Moreover, both isoflavone treatments regulated gene transcription of cytokines and inflammatory markers, among others. The transcriptomic changes provide clues firstly into defining a differential expression profile in inflammation in order to select putative biomarkers of the inflammatory process, and secondly into understanding the isoflavone action mechanism at the transcriptional level. In conclusion, isoflavone modulates the inflammatory response in activated macrophages by inhibiting NO and PGE(2) and by modulating the expression of key genes defined by transcriptomic profiling.

Metabolic adaptations to nitrogen excess in late gestation in rat.

Pregnant rats of 19th and 21st days were given an acute nitrogen overload produced by an infusion of either 0.2 M ammonium acetate or 0.2 M glutamine. Metabolic adaptations to nitrogen excess were studied measuring--in fetomaternal unit--non-protein nitrogen content and the activities of enzymes related with ammonia metabolism. Maternal and fetal plasma urea levels were increased by ammonium acetate treatment. Glutamine overload increased more the amino acid content in the mothers than in conceptus. As response to ammonium acetate treatment, glutamate dehydrogenase activity in liver was more sensitive in pregnant than in nonpregnant rats, suggesting more nitrogen incorporation into amino acids in pregnancy. Regarding glutamine synthetase activity, both treatments had an opposite effect except in kidney. The adenylate deaminase activity of pregnant rats was inhibited similarly to nonpregnant rats by nitrogen overloads, but stronger after glutamine infusion. Placenta and fetal metabolism were adjusted, as the dams, to lack of ammonia production by nitrogen overloads and to glutamine synthesis by ammonium acetate infusion.

Glutamine and ammonium handling by anaesthetized rats.

The infusion of ether anesthaetized rats with 0.2 M (1 mmols in total) ammonium acetate or glutamine were compared with the infusion of 0.2 M NaCl. The levels of circulating glucose, amino acids, lactate, urea and ammonium were measured as well as liver glycogen and tissue amino acids and the liver and muscle activities of carbamoyl phosphate synthetases I and II, glutamate dehydrogenase, glutamine synthetase and adenylate deaminase. Neither treatment altered the glucose and glycogen homeostasis. The infusion of ammonium did not result in increases in circulating ammonium, but resulted in increased circulating urea after a short delay; the infusion of glutamine resulted also in urea production but much later on. Glutamine infusion also resulted in increased tissue free amino-acid levels. There was little alteration in enzyme activities, except for decreased glutamine synthetase and adenylate deaminase activity in muscle of glutamine-infused rats and higher tissue carbamoyl phosphate synthetase II. The results agree with a fast removal of infused ammonium, and maintenance of glutamine, with their channeling towards urea production at a rate comparable with that of infusion, that did not alter significantly the homeostasis of the experimental animals.

Alterations in rat mineral metabolism induced by acute ammonium acetate infusion.

1. Acute ammonium intoxication in rat was produced by an i.v. overload of 1000 nmoles of ammonium acetate infunded during 15 min. 2. The load of ammonium produced sodium and potassium accumulation in muscle and plasma, minor in liver, and decreased these metal levels in kidney. 3. Blood and muscle magnesium content was strongly altered as a result of ammonium intoxication. 4. Calcium plasma levels, iron blood levels and iron hepatic stock diminished after the ammonium infusion. 5. Copper and zinc homeostasis were insignificantly altered.

Alpha 2-adrenergic blockade prevents hyperglycemia and hepatic glutathione depletion in nickel-injected rats.

To study the involvement of the adrenergic system in nickel-induced hyperglycemia and hepatic glutathione depletion in rats, several adrenergic antagonists (phentolamine, prazosin, yohimbine, and propranolol) were administered in a single ip injection before acute nickel treatment (ip injection). Moreover, the effects of nickel on adrenalectomized rats were investigated. Hyperglycemia was suppressed by either alpha-antagonist phentolamine or alpha 2-antagonist yohimbine. Such blockade coincided with the prevention of the hypoinsulinemic response to nickel, which occurred simultaneously to hyperglycemia. Nickel-induced hyperglucagonemia remained almost unaltered by pretreatment with adrenergic antagonists. In adrenalectomized animals treated with nickel, hyperglycemia was attenuated, whereas hypoinsulinemia still persisted. Therefore, catecholamines seemed to participate in nickel-induced hyperglycemia, directly, i.e., stimulating glucose output from liver, or by modulating insulin secretion throughout alpha 2-adrenoreceptor stimulation in pancreatic islets. Hepatic glutathione depletion caused by nickel was prevented by either alpha 1-antagonist prazosin or alpha 2-antagonist yohimbine. Interestingly, adrenalectomy did not alter the drop in hepatic GSH induced by nickel treatment. Overall results suggest that the effects observed after acute nickel exposure were caused by a combined action of catecholamines released from the adrenal glands and those released at the efferent nerves. Such events have been found to be mediated by alpha 2-adrenergic receptors.

Changes in lipolysis and hormone-sensitive lipase expression caused by procyanidins in 3T3-L1 adipocytes.

OBJECTIVE: To find out whether lipid stores are influenced by phenolic compounds in wine. DESIGN: Differentiated 3T3-L1 cells were treated with catechin, epicatechin or procyanidin extracts with different degrees of polymerization at 150 microM for different periods of time (0.5-24 h). SUBJECTS: Cell line 3T3-L1. MEASUREMENTS: Cellular viability, glycerol-3-phosphate dehydrogenase activity, glycerol release in the medium, HSL mRNA levels, triacylglycerols and protein. RESULTS: Catechin, epicatechin and procyanidin extracts were not toxic for the 3T3-L1 cells in the conditions assayed. Glycerol-3-phosphate dehydrogenase activity was markedly decreased by 150 microM procyanidin extracts. The release of glycerol into the medium was increased in 150 microM procyanidin extract-treated cells and reached a plateau after 15 h exposure. Procyanidins caused a time-dependent reduction in the HSL mRNA levels. CONCLUSIONS: These results suggest that procyanidins from grape and wine affect lipid metabolism whilst their monomers (catechin and epicatechin) do not. This effect is more pronounced when the degree of polymerization is higher. Procyanidin extracts cause a time-dependent reduction in the HSL mRNA levels, inhibit triacylglycerol synthesis and also favour triacylglycerol hydrolysis until the HSL mRNA had reached very low levels.

Amino acid metabolism in the kidneys of genetic and nutritionally obese rats.

The ability of the kidney to take up and/or release amino acids has been determined in two models of obesity in Zucker rats, one genetic and the other nutritional (diet-obese). There was a noticeable increase in gluconeogenic amino acids in the arterial blood of diet-obese animals whereas the genetically obese rats showed small variations in the levels of these amino acids. There were significant decreases in renal Gly and Ser, only in the genetically obese rats. Genetically obese animals showed an increase in Glutamine synthetase activity. The uptake and/or release of amino acids showed important variations between the groups. The diet-obese group exhibited greater variation, since this group took up Glu, Ala, Gy, Phe and Citrulline and released Gln, Ser, Arg and Tyr. Genetically obese rats took up Gln, His and Taurine and released Ser. These different patterns may be related to variations in the whole body metabolic rate, since the diet-obese group was more active than the genetically obese group.

Muscle amino acid pattern in obese rats.

OBJECTIVE: To determine how the ability of skeletal muscle to manage amino acids is conditioned by obesity. The test was performed in two different models of obese rats: diet-obese rats and genetically obese rats. SUBJECTS: Lean and genetically obese (fa/fa) male Zucker rats were used. DESIGN: For up to 60 d of life lean animals were fed with standard chow pellet or with a hypercaloric cafeteria diet. Genetically obese rats were fed with standard chow pellet during the same period. MEASUREMENTS: Amino acid concentration in the femoral artery and vein and leg blood flow were measured. Free amino acid concentration, protein and nitrogen content and enzyme activities were determined in whole leg muscle. RESULTS: Amino acid availability was increased in diet-obese animals and remained unchanged in the genetically obese group. The genetically obese groups had a lower protein content and unchanged concentration of amino acids in leg muscle. Furthermore, total nitrogen remained unchanged in these animals and they showed an increased activity in alanine aminotransferase and glutamine synthetase. The leg muscle of the diet-obese rats took up several amino acids (Ala, Arg and Val) or released others (mainly Gln and Gly). Conversely, genetically obese rats took up many amino acids and did not release any. CONCLUSIONS: We conclude that in nutritionally obese rats there is an increased availability of amino acids in skeletal muscle. This augmented availability propitiates the increase in the uptake of many amino acids. In genetically obese rats, the lack of variation in amino acid availability points to a possible hereditary alteration that increases the capacity of different amino acid transport systems. Furthermore, the diminished protein content of fa/fa muscle is not due to a lower availability and intracellular pool of amino acids.

Effects of copper exposure upon nitrogen metabolism in tissue cultured Vitis vinifera.

The present study analyses the effects of copper treatment on nitrogen metabolism in a closed system. Sauvignon grapevines were cultured in agar and exposed to copper levels ranging from 0.07 (control) to 5 µg Cu g(-1) medium. Ammonium, nitrate, nitrite, individual and total amino acids and protein contents were determined in root, and leaves. Enzyme activities of nitrate and nitrite reductases, glutamine synthetase, glutamate synthase and glutamate dehydrogenase were also determined. Copper exposure produces a dramatic change in nitrogen metabolism, with a reduction of total nitrogen, which reflects the reduction on nitrate and free amino acid contents in both root and leaves. The assimilation of nitrate the main nitrogen source in the medium, requires nitrate reductase, which is reduced to negligible activity as response to copper exposure. Primary nitrogen metabolism is also reduced in leaves, although to a lesser extent than in roots, which may explain the differences between the two organs in response to copper exposure. An alternative system for assimilation of nitrogen through glutamate dehydrogenase in roots is proposed, while higher levels of ammonium and glutamine may fullfil the needs of organic nitrogen in the leaves.

Splanchnic amino acid pattern in genetic and dietary obesity in the rat.

The study of intestinal and hepatic uptake of amino acids by obese rats has been the main objective of this work. The obese animals used were either from genetic or from nutritional basis. In fed state, the intestinal release of amino acids was higher in obese animals than in lean ones (around the double values), but nutritionally and genetically obese rat showed a related pattern, specially for the case of alanine (increased release in relation to controls by a factor of 10). The higher alanine release by intestine is not reversed by 12-h food deprivation. The hepatic availability was also higher in obesity models than in lean animals (increases over 30%). However, the hepatic uptake was increased in genetically obese animals (more than 35%) and decreased in nutritionally obese animals (more than 40%), especially due to alanine uptake (2419, 1100 and 3794 nmols/min/g protein in lean, Diet-ob and fa/fa animals respectively). In obese animals the food deprivation tended to normalize the hepatic uptake of alanine. The differences in alanine uptake between both types of obesity may reflect the differences of urea synthesis.