Influence of interleukin-1alpha and COX-2 over the metabolism of arachidonic acid and glucose in isolated uterus of restricted diet rats.

Isolated uteri from rats fed with a normal diet convert [14C]arachidonate into eicosanoids: PGE(2), PGF(2alpha), TXB(2) and 6-keto-F(1alpha). Restricted diet (50% of the normal diet, during 25 days) diminishes the levels of PGE(2), PGF(2alpha) and TXB(2). The addition of Interleukin-1alpha to the Krebs-Ringer bicarbonate medium increases sharply the production of eicosanoids. Inhibitors of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester or aminoguanidine, do not prevent eicosanoids increase. Conversely, NS-398 (a selective inhibitor of COX-2) blocks the increase of eicosanoids while PGE(2) blocks eicosanoids production mediated by IL-1alpha. Other experiments with uteri of underfed rats confirm that interleukin-1alpha produces an increase in the glucose metabolism. The addition of Nomega-nitro-L-arginine methyl ester, aminoguadinine or NS-398 blocked such stimulation. It is concluded that Interleukin-1alpha produces an increase of glucose metabolism in uteri isolated from underfed rats by two different mechanisms, both involving COX-2: (1) nitric oxide independent and (2) nitric oxide dependent.

Effect of interleukin 1alpha and interleukin 2 over glucose metabolism in isolated uterus of restricted diet rats. Influence of NO and COX-2.

A 25-day dietary restriction (50% of the normal diet) produce a fall in the production of 14CO2 from 14C-glucose in rats isolated uteri. The addition of 10 or 20 ngml(-1) interleukin 1alpha (IL-1alpha) or interleukin 2(IL-2) to the Krebs-Ringer bicarbonate solution medium stimulates glucose metabolism in uteri from underfed rats. Such concentrations are not effective in control rats. The addition of Nomega-nitro-L arginine methyl ester--an inhibitor of both the constitutive and inducible forms of nitric oxide synthase (NOS)--and of aminoguadinine--a preferential inhibitor of the inducible form of NOS--block such stimulation. In other experiments, the addition to the medium of arginine-a substrate for the formation of nitric oxide-increases interleukin stimulation of glucose metabolism, which is blocked by NOS inhibitor. At the same time, NS-398--a selective inhibitor of inducible cyclooxygenase (COX)--eliminates the interleukin metabolism stimulation. We conclude that IL-1alpha and IL-2 produce an increase of glucose metabolism in uteri isolated from underfed rats. Nitric oxide produced by the inducible form of NOS mediates the interleukins-induced glucose metabolism stimulation with the participation of inducible COX.

Opioid effects on glucose and eicosanoid metabolism in isolated uterus of ovariectomized and non-ovariectomized restricted diet rats.

The effect of a 25-day restricted diet (50% of the normal food intake) on uterine glucose metabolism of ovariectomized (25 days) and non-ovariectomized rats, was studied. Underfeeding reduces (14)CO(2) production from U(14)C-glucose in intact animal. However, in spayed rats, results are the opposite. In intact rats receiving a low food intake, the effect of the addition to the KRB medium of various agonist opioids, was studied. Dinorphin A did not bring about any change. On the other hand, beta endorphin increased glucose metabolism. Also, the addition of Dago and Dadle increased (14)CO(2) production, while their corresponding specific blockers, beta-FNA and Naltrindole, reversed it. Ovariectomized rats subjected to food restriction are not affected by opioid agonists. In vitro morphine, like endogenous opioids, increased (14)CO(2) in intact restricted diet rats. Arachidonic acid metabolism in these rats show that underfeeding brings about a decrease in PGF(2 alpha) and PGE(2), but the addition of morphine does not alter this situation, for which eicosanoids metabolites are not related to the effect of morphine. The morphine effect was not altered by naloxone. The subcutaneous injection of morphine increased glucose metabolism in intact underfed animals, while naloxone reduced (14)CO(2) in spayed rats subjected to underfeeding. It can be concluded that uteri from ovariectomized rats receiving a restricted diet are influenced by a mechanism of upregulation related to endogenous opioids. These likely originate in other tissues, and so prevent us from seeing the morphine effect.

Arginine degradation by arginase in mitochondria of soybean seedling cotyledons.

Arginase (EC 3.5.3.1) localization was studied in soybean (Glycine max L.) seedling cotyledons. Subcellular fractionation in a discontinuous Percoll gradient showed that arginase was localized in the mitochondrion. Arginine (Arg) uptake by mitochondria was demonstrated by co-sedimentation of [3H]Arg-derived label and the mitochondrial marker enzyme cytochrome c oxidase. Arginine uptake was complete in about 10 min. Since detergent but not NaCl released most label, we conclude that Arg was taken up and not bound to the organellar surface. Arginine transport was not saturable, at least up to 20 mM. Basic amino acids were the best inhibitors of Arg uptake. The uncoupler 2,4-dinitrophenol did not inhibit Arg uptake. At least 30% of L-[guanido-14C]Arg taken up by mitochondria was degraded by arginase in seedling cotyledons, while little or no degradation was detected in mitochondria from developing embryos, even though the Arg uptake level was similar in both mitochondrial preparations. These results are consistent with our previously reported pattern of arginase expression and urea accumulation during embryo development and seed germination (A. Goldraij and J.C. Polacco, 1999, Plant Physiol. 119: 297-303). The lack of Arg degradation allows developing embryos to conserve Arg, the main N-reserve amino acid utilized by germinating soybean.

Effects of morphine and naloxone on glucose metabolism in uterine strips from ovariectomized and non-ovariectomized restricted diet rats.

The effect of underfeeding over glucose metabolism in uteri isolated from ovariectomized and non-ovariectomized rats subjected to a restricted diet for 25 days (50% of the normal food intake), was studied. Underfeeding decreases (14)CO(2) formation from U(14) C-glucose in intact animal uteri. While in ovariectomized rats (25 days), the effect is the opposite. The addition of morphine 10(-6) M to the medium does not affect rats fed ad libitum. However, (14)CO(2) levels increase significantly in intact animals receiving a restricted diet. In ovariectomized rats morphine does not show any activity, regardless of the type of diet rats were subjected to. None of the rat groups seems to be sensitive to naloxone 10(-6) M. The s.c. injection of morphine (4 mg.kg (-1)) increases glucose metabolism only in intact rats provided with a restricted diet, while naloxone (2.5 mg.kg (-1) ) produces a decrease of ( 14)CO(2) in ovariectomized underfed animals. To conclude, morphine either 'in vivo' or 'in vitro' is active only in uteri from intact rats subjected to underfeeding. Naloxone produces a decrease in (14)CO(2) production, particularly when it is s.c. injected to ovariectomized rats undergoing a dietary restriction. Since the uterus does not react to naloxone, the effect of the opiod blocker may be the result of endogenous opioids originated in other tissues.

Influence of insulin on the metabolism of glucose in uteri isolated from ovariectomized and non ovariectomized underfed rats.

The effects of insulin on the metabolism of U14C-glucose in uteri isolated from ovariectomized and non-ovariectomized rats receiving a restricted diet (50% of the normal food intake) for 25 days, were studied. As a result of food restriction, the production of 14CO2 diminishes in intact rats, while results are reversed in ovariectomized ones. Various concentrations of insulin were added to the medium, but only 0.50 IU. ml(-1)was effective in increasing glucose metabolism in intact rats receiving a restricted diet; neither underfed castrated animals nor control ones receiving a normal diet, reacted to this concentration. The increase of 14CO2 produced by insulin is not affected by acetyl salicylic acid. Insulin does not alter the effect of underfeeding over arachidonic acid metabolism. On the contrary, the increase in glucose metabolism was blocked by N(G)methyl-L-arginine or by hemoglobin, increased with the addition of L arginine and is not affected by acetyl salicylic acid. Hemoglobin and L-arginine show no effects without insulin. We can conclude that the stimulating effect of insulin on glucose metabolism in uteri isolated from intact rats subjected to dietary restriction, is nitric oxide dependent.

Arginase is inoperative in developing soybean embryos.

Arginase (EC 3.5.3.1) transcript level and activity were measured in soybean (Glycine max L.) embryos from the reserve deposition stage to postgermination. Using a cDNA probe for a small soybean arginase gene family, no transcript was detected in developing embryos. However, arginase transcripts increased sharply on germination, reaching a maximum at 3 to 5 d after germination. There was low but measurable in vitro arginase specific activity in developing embryos (less than 6% of seedling maximum). During germination arginase specific activity increased in parallel with the sharply increasing arginase transcript level. Seedling arginase activity was largely localized in cotyledons. Arginase activity was assayed in vivo by measuring urea accumulation in a urease-deficient mutant. No urea was detected in developing embryos, whereas accumulated urea paralleled arginase specific activity and transcript level in germinating seedlings. As in planta embryos, cultured cotyledons did not accumulate urea when arginine (Arg) was provided with other amino acids in a "mock" seed-coat exudate. Arg as the sole nitrogen source was converted to urea but did not support cotyledon growth. There appeared to be a lack of recruitment of the low-level arginase activity to hydrolyze free Arg in developing embryos, thus avoiding a futile urea cycle.

Effect of a restricted diet on the metabolism of glucose in uteri isolated from immature rats: influence of indomethacin and nordihydroguaiaretic acid.

In immature female rats (21 days), a restricted diet (50% of the daily normal intake for 25 days) interrupts sexual development, leaving animals in a state of sexual immaturity. Food restriction does not affect the use of glycogen in uteri isolated and increases 14CO2 production from U14C-glucose in relation to animals receiving normal feeding that reach sexual maturity. Indomethacin and acetylsalicylic acid stop the increase of glucose metabolism produced by underfeeding, without affecting the uteri of rats receiving normal feeding. The addition of PGE2 and PGF2alpha changes the inhibitory effect of indomethacin. Nordihydroguaiaretic acid produces the opposite effect, increasing glucose metabolism only in uteri isolated from immature animals. These results show that immature animals have a higher glucose metabolism if compared to mature rats. The restricted diet, which slows down sexual maturity, keeps this parameter high due to the influence of some eicosanoids.

Effects of inhibitors of nitric oxide synthase on isolated uteri of fasting rats.

The effects of inhibitors of nitric oxide synthase on the glucose metabolism of uteri isolated from 4-day underfed rats were studied. In control rats receiving normal feeding, the addition of indomethacin (5 x 10(-6) M); acetyl salicylic acid (10(-4) M); 400 microM of N(G)methyl-L-arginine, (L-NMMA) or 400 microM of sodium nitroprusside (SNP), does not modify the production of 14CO2 from U14C-glucose. On the contrary, in fasted rat uteri, indomethacin increases glucose oxidation significantly, while acetyl salicylic acid does not alter it. Also, the addition of L-NMMA has no effect. In another group of experiments, in the preparations containing indomethacin of uteri isolated from underfed rats, the addition of L-NMMA significantly changes the effect of indomethacin. Another inhibitor of nitric oxide synthase, N(omega)nitro-L-arginine methyl ester (L-NAME), or hemoglobin (2 microg ml(-1)) a nitric oxide scavenger have the same effects while N(omega)nitro arginine-D-methyl ester (D-NAME) does not. However (SNP), a nitric oxide donor, does not alter the production of 14CO2 in uteri isolated from fasted rats. These results show that in underfed rats, indomethacin increases glucose oxidation independently from its inhibiting effect on cyclooxygenase. Specific inhibitors of nitric oxide synthase can reverse this effect.

Purification of rabbit skeletal muscle proteoglycogen: studies on the glucosyltransferase activity of polysaccharide-free and -bound glycogenin.

Proteoglycogen is the end product in the process of glycogen biogenesis. We have purified rabbit muscle proteoglycogen and studied the glucosyltransferase reactions catalyzed by its protein moiety, glycogenin, free or bound to the polysaccharide. The purification strategy involved dissolution of proteoglycogen and cosedimenting membrane vesicles in a Triton X-114/Triton X-45 mixture followed by partition in the aqueous phase, potassium iodide precipitation of accompanying proteins, and washing by high-speed centrifugation. Glycogenin or a proteoglycogen species of an average molecular mass of 200 kDa was isolated by ion-exchange chromatography after the purified proteoglycogen had been subjected to long or short amylolytic digestion, respectively. Besides autoglucosylation from UDP-glucose, glycogenin was capable of autogalactosylation from UDP-galactose. The autoglucosylation reaction was not inhibited by the simultaneous glucosylation of the exogenous acceptors N-(maltosyl-alpha-1-4-(1-deoxiglucitol))-peptide or n-dodecyl-beta-D-maltoside. The polysaccharide-bound glycogenin species of 200 kDa showed to be active for the glucosylation of exogenous acceptor and represented the isolated proteoglycogen of higher size having glucosyl transferase activity. This is the first description of the isolation of native proteoglycogen and a proteoglycogen species having glucosyltransferase activity.

Effects of 17 beta estradiol on the metabolism of labelled arachidonic acid in uteri isolated from intact and ovariectomized rats. Influence of a restricted diet.

The effects of restricted diet (50% of the normal intake during 25 days) on the metabolism of 14U arachidonic acid, were explored in uterine horn strips isolated from intact and ovariectomized rats, treated by 17 beta-estradiol or controls. The metabolism of arachidonic acid into different eicosanoids, PGE2, PGF2alpha, 6-keto PGF1alpha and TXB2, showed that the restricted diet diminished PGE2 and PGF2alpha, in intact rats, significantly. In contrast, this kind of feeding did not produce any change in castrated rats. Tissue preparations from previously estrogenized intact and castrated normal-fed rats showed that the production of different metabolites decreased. A similar result was obtained in intact rats subjected to a restricted diet. Nevertheless, in castrated underfed rats, estrogens did not produce any effect on the various eicosanoids analysed. These results showed that in isolated uteri, the effects of 17 beta-estradiol, on metabolite production from labelled arachidonic acid, are different from controls in ovariectomized diet-restricted rats.

Effect of fasting on the contractile activity and metabolism of labelled glucose and arachidonic acid in uteri isolated from intact and ovariectomized rat.

The effects of fasting for 4 days on the isometric developed tension (IDT) and on the metabolism of labelled glucose and arachidonic acid in uteri from intact and spayed (25 days) rats, were explored. Starvation produces a fall in the contractile activity of intact rats, while in ovariectomized ones, no differences can be seen with respect to their controls. Fasting produces a fall in the glucose metabolism of both intact and ovariectomized rats, being more noticeable in the former group. Indomethacin (5 x 10(-6) M) increases the metabolism of labelled glucose in all experimental groups, significantly. The metabolism of exogenous arachidonic acid into different eicosanoids, PGE2, PGF2 alpha, 6-keto-F1 alpha and TXB2, shows that total food deprivation diminishes significantly the production of PGE2 in intact rats. In contrast, in ovariectomized starved rats, PGE2 increases markedly. The rest of the metabolites studied are not influenced by fasting. These results show that the effects of fasting on the contractile activity and on the release of some metabolites from arachidonic acid by the uteri isolated from intact rats are not seen in ovariectomized animals.

M-glycogenin, the protein moiety of Neurospora crassa proteoglycogen, is an auto- and transglucosylating enzyme.

Neurospora crassa proteoglycogen was purified and its protein moiety, M-glycogenin, was released by amylolytic treatment. The released protein was capable of autoglucosylation from UDP-glucose forming glucosyl-alpha 1,4-glucosyl linkage. The kinetics of autoglucosylation suggested an intramolecular mechanism of reaction. M-glycogenin was also able to glucosylate dodecyl-beta-maltoside and autoglucosylate, simultaneously and independently. Both auto- and transglucosylation reactions were dependent on Mn2+. Thus, M-glycogenin, which has also been described as the constituent of Escherichia coli proteoglycogen (A. Goldraij and J. A. Curtino. 1993, Biochem. Mol. Biol. Int. 30, 453-458), is a glucosyltransferase that bears similar catalytic properties with mammalian glycogenin. This is the first report on the enzymatic character of the protein constituent of proteoglycogen in primitive organisms, which suggest that the mechanism for the de novo biosynthesis of glycogen was conserved over a very long period of evolution.

Influence of underfeeding on the spontaneous contraction and on metabolism of labelled arachidonic acid in uteri isolated from intact and ovariectomized rats.

The effects of restricted diet (50% of the normal intake during 25 days) on the isometric developed tension (IDT), and on the metabolism of labelled arachidonic acid in uteri from intact and spayed (25 days) rats, were studied. Underfeeding produced a fall in the contractile activity of intact rats, while in ovariectomized rats contractile activity increased. Indomethacin reduced uterine contractile activity in all the cases under study. The metabolism of arachidonic acid into different eicosanoids, PGE2, PGF2 alpha, 6-keto-F1 alpha, and TXB2, showed that the restricted diet diminished PGE2 and PGF2 alpha levels in intact rats significantly. The reduction in uterine contractile activity and reduced levels of the arachidonic acid metabolites, PGE2 and PGF2 alpha, were not seen in ovariectomized animals.

Effects of ovarian steroids on glucose metabolism in uterus horns isolated from underfed rats. Influence of castration.

The effect of a restricted diet (50% of the normal intake) during 25 days, on the glucose metabolism was explored in uterus horn strips isolated from intact and ovariectomized rats. In intact underfed rats, the formation of 14CO2 from U14C-glucose was significantly lower than in their controls. In castrated rats, the formation of 14CO2 increased after underfeeding. The return to ad libitum feeding increased glucose metabolism in both groups. Total food deprivation for 4 days diminished 14CO2 formation both in non-ovariectomized rats and in ovariectomized ones as measured at the end of this fasting period. This diminution was greater in intact rats. The increase of glucose metabolism provoked by the administration of s.c. estradiol, progesterone or their combination was more important in underfed intact animals. In intact rats, blood levels of endogenous estradiol and progesterone decreased as a result of underfeeding. In spayed ones, their low concentration increased in relation to their controls.

Different effect of a restricted diet on isolated uteri of ovariectomized and non-ovariectomized rats. Influence of indomethacin and prostaglandins.

The effect of restricted diet (50% of the normal intake during 25 days) on the metabolism of labelled glucose, in uteri isolated from ovariectomized (25 days) and non-ovariectomized rats, was explored. In intact rats subjected to dietary restriction, the formation of 14CO2 from U 14C-glucose is significantly lower than in controls. Indomethacin increases glucose metabolism, being even higher in underfed rats' uteri. This effect is not altered by the addition of prostaglandins E1, E2 or F2 alpha to the medium. In castrated rats, the formation of 14CO2 increases due to underfeeding and this result is not altered by indomethacin. Glycogen and triglyceride values in the isolated uterus were measured immediately after killing (O time) and after 60 min of incubation in a glucose-free KRB medium. The post-incubation levels of glycogen from intact normal fed animals diminished in comparison to initial values, and this result was not altered by the addition of indomethacin. In rats subjected to dietary restriction, glycogen did not decline further after incubation, and the addition of indomethacin led to a significant fall. In spayed rats, glycogen diminished after 60 min both in normal fed rats and in underfed ones, and they were not affected by the indomethacin. In intact underfed rats, uterine triglycerides fall after 60 min. Indomethacin changes this situation, which is again evoked by the addition of PGF2 alpha. In ovariectomized rats, uterine triglycerides are neither altered by a restricted diet nor affected by indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a restricted diet on in vitro spontaneous activity and glucose metabolism in isolated rat uterus. Influence of castration.

The effects of a restricted-diet (50% of the normal intake during 25 d) on the isometric developed tension (IDT), the metabolism of labelled glucose, and the levels of glycogen, of uteri isolated from ovariectomized (25 d) and non-ovariectomized rats were explored. The restriction of food intake produced a fall in the contractile activity of normal, non-ovariectomized, rats in permanent diestrous compared to normally fed rats in diestrous. On the contrary, in castrated rats, the IDT of isolated uterus from underfed rats, was significantly higher than its normal-fed controls. In normal rats the formation of 14CO2 from U 14C-glucose was significantly lower in uterine preparations from restricted-diet animals than the control one. On the other hand, in castrated rats, the formation of 14CO2 increased as a result of underfeeding. The post-incubation levels of glycogen in uteri from normal-fed animals diminished significantly in comparison to 0 time values. In uteri from rats subjected to a dietary restriction, the initial glycogen values were lower than in normal-fed controls, but they did not decline further after incubation in KRB medium. On the contrary, even when the levels of glycogen were significantly lower at 0 time than in diestrous animals, they diminished in ovariectomized rats after incubation, no matter the diet. The above results indicate that the effects of restricted-diet on contractile activity, levels of glycogen and glucose metabolism were not observed in ovariectomized rats. Further researches are needed to clarify that point.

Glycogen-bound protein in lower eukaryote and prokaryote.

The proteoglycogen fraction of Neurospora crassa was purified and subjected to radioiodination with [125I]iodide. Amylolysis of the polysaccharide moiety led to the isolation of a labelled 31 kDa-protein. The NH2-terminal amino acid sequence of 10 residues of the 31 kDa-protein was determined. A 31 kDa-protein was also bound to glycogen in Escherichia coli. Proteoglycogen has not been heretofore found in any primitive unicellular organism.

Effect of chronic underfeeding on uterine glycogen of rats. Influence of indomethacin and nordihydroguaiaretic acid.

Glycogen values in uterine strips isolated from normal-fed estrous or diestrous rats, or from rats fed a restricted diet (50% of normal food intake for 25 days) were measured. Determinations were made immediately after killing (0 time or post-isolation) as well as after incubation in glucose-free medium (60 min time or post-incubation). The post-incubation levels of glycogen in the uteri from normal-fed animals diminished significantly in comparison to post-isolation values, and this decrement was not modified by the addition of indomethacin, nordihydroguaiaretic acid or exogenous prostaglandins E1, E2 or F2 alpha. In rats fed a restricted-diet, the initial glycogen values (0 time) were significantly lower than in normal-fed controls, but did not decline further after incubation in glucose-free medium (60 min time). The addition of indomethacin, acetylsalicylic acid or of nordihydroguaiaretic acid led to a significant fall in the glycogen levels, and exogenous PGE1, PGE2 or PGF2 alpha failed to alter the effects of the inhibitors. The values of PGE and PGF prostaglandins release to the medium by the uterus from restricted-diet rats did not differ from those obtained in the experiments with normal-fed animals. Administration of 17-beta estradiol to restricted-diet rats led to suppression of the effects of this diet on the glycogen concentration. The above results indicate that in rats subjected to a prolonged period of dietary restriction, the uterine glycogen becomes responsive to the effects of cyclooxygenase and lipoxygenase inhibitors, suggesting the operation of some regulatory mechanism during critical periods of nutrition.

Spontaneous activity in vitro of the uterine horns of unilaterally pregnant rats. Relations with glycogen and triglycerides levels.

Effects of the placental implantation on the in vitro spontaneous contractile activity of uterine strips incubated in a glucose-free KRB medium, and its relationship with the glycogen and triglycerides tissue levels, have been analysed using unilaterally pregnant rats. The spontaneous activity increased with pregnancy duration, both in the implantation zone (Impl) and the interembryonic segment (Inter) of the pregnant horn. It increased in the contralateral sterile horn (SH) also. Activity was significantly greater in SH and Inter than in Impl, at the same stage of pregnancy. Uterine glycogen, but not triglycerides, appeared to be the substrate used to sustain contractile activity, as its concentration was greater in Impl, the relatively quiescent zone.