Browsing by Author "Zorzano, Antonio"

The dose-response relationship in brain. plasma, and adrenal mon()aminc changes after acute oral ethanol administration (I, 2. 4 g./kg body wt) was sl u-licd in virgin rats lo determine whether the response lo the highest dose differed in 21-day pregnant ani11ials. and to assess the potential consequences of ethanol on the neurotransmitlcr systems of their fctusc s. Blood ethanol and acetaldchyde concentrations in blood increased progrcs:;ivcly with the ctha1wl dose in virgin rats, and values in pregnant animals were very similar. Ethan,>! concentration in fetal blood and amniotic Ouid did not differ from that in mother's blood whereas fetal acctaldchydc concentrations were negligible. In a dose-related manner, ethanol decreased brain DA. DOPAC and 5HT ..:cncentrations did not affect those of NA and 5HIAA, or adrenal A and NA concentrations, whereas it enhanced plasma NA levels. Basal levels of monoamincs and their changes after ethanol intake did not differ in pregnant and virgin rats. Monoaminc and metabolite concentrations were much lower in fetal tlian in maternal brains whereas plasma and adrenal catecholamine concentrations were very similar and naternal ethanol intake did not modify these fetal parameters in the fetus. Results are in agreement wi1 h the known similar metabolic response to ethanol in fed pregnant and virgin rats. The lack of fetal monoamine response to maternal ethanol intake may be a consequence of the incapacity of fetal liver to form acetaldchydc and the ability of the placenta to oxidize maternal acctaldehyde which protects the fetus fr,m1 maternal alcohol intake at late gestation.

The rediscovery of brown adipose tissue (BAT) in humans and its capacity to oxidize fat and dissipate energy as heat has put the spotlight on its potential as a therapeutic target in the treatment of several metabolic conditions including obesity and diabetes. To date the measurement of bioenergetics parameters has required the use of cultured cells or extracted mitochondria with the corresponding loss of information in the tissue context. Herein, we present a method to quantify mitochondrial bioenergetics directly in BAT. Based on XF Seahorse Technology, we assessed the appropriate weight of the explants, the exact concentration of each inhibitor in the reaction, and the specific incubation time to optimize bioenergetics measurements. Our results show that BAT basal oxygen consumption is mostly due to proton leak. In addition, BAT presents higher basal oxygen consumption than white adipose tissue and a positive response to b-adrenergic stimulation. Considering the whole tissue and not just subcellular populations is a direct approach that provides a realistic view of physiological respiration. In addition, it can be adapted to analyze the effect of potential activators of thermogenesis, or to assess the use of fatty acids or glucose as a source of energy.

Reviews these carbohydrate-lipid interactions and analyzes the role of maternal body fat accumulation during the early part of pregnancy in the metabolic adaptations during late gestation. The consequences and intimately related aspects of these interactions are several of the other changes that occur in the maternal/fetal relationship such as the· development of maternal hypertriglyceridemia, changes in the maternal amino acid metabolism, placental metabolite transfer, etc. These topics are reviewed in the other chapters of this book.

-I. The appearance of plasma [ 14C]glucose in the inferior cava vein after a pulse of 0.2 mmol of [U- 14C]L-alanine or [U-14C]glycerol/200 g body wt given through the portal vein was studied in fed 21 day pregnant rats and virgin controls under pentobarbital anesthesia. In both groups values were much higher when [U- 14C]glycerol was the administered tracer than when [U- 14C]L-alanine, and they were augmented in pregnant versus virgin animals at 1 min when receiving [U- 14C]glycerol and at 2 min when receiving [U- 14C]L-alanine. 2. 20 min after the tracers rats receiving (U- 14C]glycerol showed much higher liver ( 14C]g]ycogen and [ 14C]glyceride glycerol than those receiving [U- 14C]L-alanine. Radioactivity present in liver as [1 4C]glyceride glycerol was greater in pregnant than in virgin rats receiving [U- 14C]glycerol whereas radioactivity corresponding to [ 14C]fatty acids was lower in the former group receiving either tracer. 3. At 20 min after maternal treatments fetuses showed lower plasma [' 4 C]glycerol than [ 14C]alanine values but plasma [ 14C]glucose and liver [ 14C]glycogen values were much greater in fetuses from mothers receiving [U- 14C]glycerol than (U- 14C]L-alanine. 4. Besides showing the higher gluconeogenic efficiency in pregnant than in virgin rats, results indicate that at late gestation glycerol is used as a preferential substrate for both glucose and glyceride glycerol synthesis in liver.

I. On the basis of kinetic properties and sensitivity to pyrazole inhibition, it is shown that li~er alcohol dehydrogenase present in human mainly corresponded to class I and in rat to class ADH-3 which differed in a number of parameters. _ 2. Two different aldehyde dehydrogenase (ALDH) isoenzymes were detected m both human and rat liver. The human isoenzymes corresponded to the ALDH-1 and ALDH-11 trpe. _ _ 3. In the rat one isoenzyme had low Km and showed similar activity than m human hver ~ut differed in their sensiti;ity to both disulfiran and nitrofazole inhibition whereas the other presented high Km and showed greater activity than the human one. 4. Caution must be therefore paid when extrapolating to human subjects the data on ethanol metabolism obtained with rats.

The characteristics of the major human hepatic isoenzymes of aldehyde dehydrogenase (ALDH), ALDH I and ALDH II, were compared with the ALDH activities found in human placenta and erythrocytes. (ii) In human liver biopsies, the Km of ALDH I was approximately 7 μmol/L whereas it was 32 μmol/L for ALDH II. The V max for ALDH I was 2-3 times greater than the ALDH II V max· Human liver ALDH I and II also differed in their sensitivity in inhibitors. Namely, ALDH I was less sensitive to disulfiram than the ALDH II isoenzyme. (iii) ALDH activity in human placenta and erythrocytes was much lower than in liver tissue. Kinetic data showed that placental ALDH isoenzyme had a high Km (in the millimolar range) and increased its activity raising the pH from 7.4 to 8.8, more than the hepatic ALDH I and ALDH II isoenzymes did. Erythrocyte ALDH activity presented a dual component; the smaller one was characterized by a low Km (micromolar range), whereas most of the ALDH activity showed a high Km (millimolar range). (iv) Placental ALDH was resistant to nitrefazole inhibition and was inhibited by disulfiram in a manner similar to the hepatic ALDH I isoenzyme; erythrocyte ALDH was more sensitive to the inhibitory action of disulfiram and nitrefazole. (v) It is concluded that erythrocyte and placental ALDH isoenzymes are different from the hepatic ALDH I and ALDH II forms. It is also suggested that placental and erythrocyte ALDH isoenzymes are different high-Km isoenzymes.

The pattern of ethanol and acetaldehyde appearance in blood after ari oral ethanol gavage (4 g/kg body wt) was not different at 12 or 21 days' gestation compared to virgin rats. Five min after maternal ethanol administration, concentrations of ethanol in fetal blood were lower than in maternal blood; however, at 15 min after ethanol administration, fetal and maternal blood levels were similar. Ethanol concentrations in fetal blood and amniotic fluid were already at equilibrium 5 min after ethanol administration. Acetaldehyde concentrations in fetal blood and in amniotic fluid were undetectable at all the times investigated, with the exception of fetuses from two pregnant rats studied 3 h after ethanol administration. Alcohol dehydrogenase activity in fetal liver and in placenta at late pregnancy was very low or undetectable, suggesting a very low rate of ethanol oxidation in vivo. After intravenous administration of acetaldehyde (10 mg/kg body wt), blood acetaldehyde concentrations were higher in pregnant than in virgin rats. Acetaldehyde concentrations in fetal blood and in amniotic fluid were similar to maternal blood at 2, 5, and 30 min after injection. When circulating concentrations of maternal and fetal acetaldehyde, obtained after either ethanol or acetaldehyde administration, were plotted, it was found that fetal blood concentrations of acetaldehyde were only detectable when maternal blood concentrations were greater than 80 µM. Concerning the acetaldehyde oxidation capacity, both the high and low affinity components of aldehyde dehydrogenase activity in fetal liver and placenta were very low as compared with maternal liver. However, aldehyde dehydrogenase activity in fetal liver was much higher than in placenta. It is proposed that ethanol present in the feto/placental unit is metabolized mainly by the maternal liver and that there is a threshold for acetaldehyde above which the capacity for acetaldehyde metabolism is surpassed.

Ethanol is transformed in the mother by the catalytic action of alcohol and acetaldehyde-dehydrogenases, and the products of these reactions (NADH and acetaldehyde) are responsable of most of the negative effects of ethanol. Ethanol crosses the placenta freely, reaching in the feta! circuk.tion the same levels as in the mother. The placenta has the capacity to oxidyze acetaldehyde, thus maternal acetaldehyde does not reach the fetus. Besides this, the fetus does not have etha11ol metabolyzing enzymes. By using the rat as experimental model, we have found that metabolites known to cross the placenta such as glucose and ketone bodies vary in the fetus in a parallel manner as in the mother after alcohol intake. However, compounds known not to cross the placenta such as triglycerides do not change in the fetus whereas they increase in both plasma and liver of the mother after alcohol intake. After birth, retarded development of newborns from mothers receiving alcohol throughout gestation is progressivelly recuperated, but this recuperation depends on the parameter studied and the nutritional and environmental condition. The animal model herein used is valid for studies on the feta! alcohol syndrome, and although extrapolation to humans must be done with caution. present results indicate that most negative effects of maternal alcohol ingestion on the fetus are secondarv to ethanol action on maternal metabolism. and the\' mav be ameliorated by an adequate postnatal nutritional condition.

Our data demonstrate that (a) marked modifications in ADH activity, as found in patients with atypical ADH or in subjects with alcoholic liver disease, are not accompanied by parallel alterations in the kinetics of ethanol disappearance, suggesting that ADH activity per se does not limit ethanol metabolism in viva, (b) hepatic high-Km ALDH activity is decreased in patients with liver disease independent of alcoholism, and therefore decreased ALDH activity cannot be considered as a primary defect in alcoholism but as a consequence of liver damage. and (c) erythrocyte ALOH does not reflect hepatic high-Km ALOH.

To study in vivo gluconeogenesis, female virgin rats ere injected intravenously with ,.C-alanine (ul) and the production of 14C-glucose was determined at two. five, or ten min tes thereafter. At ten minutes the appearance of 14C-glycogen in the liver was also determined. The intraperitoneal inje tion of sodium pentobarbital (Nembutal) (33 mg/kg body weight) 30 minutes prior the tracer did not affect the rate of gluconeogenesis in fed rats compared with unanesthetized animals, whereas in rats fasted 24 hours it produced a si nificant enhancement in all parameters studied. A similar effect in enhancing in vivo gluconeogenesis was observed ith both pentobarbital or ether anesthesia when 3-14C-pyruvate was used as tracer in virgin rats fasted 24 hours. In con rast to the effect in virgin animals, pentobarbital anesthesia did not modify in vivo gluconeogenesis in either fed or 24-h ur fasted 21-day pregnant rats. Ether anesthesia, however, caused an enhancement in "C-glucose production from 3- 14C pyruvate in 24-hour fasted pregnant rats. On the basis of reported changes in sympathoadrenal activity produced by s arvation and pregnancy, present results indicate that the enhancing effects of anesthetics on gluconeogenesis result fro their capacity to stimulate adrenal medulla cathecholamine release or tissue sympathetic activity. Our findings also dem nstrate that in an investigation of metabolic parameters it cannot be assumed that effects of anesthetics are always of th same degree and direction since they vary with the condition of the experimental subject.

In the late pregnant rat, blood glucose levels were lower and plasma RIA-insulin levels were slightly higher than in virgin animals. Oral and intravenous glucose tolerance tests produced parallel changes in blood glucose in both groups whereas plasma RIA-insulin increased more in the pregnant animals. Blood glucose levels after either low (0.1-1 IU/Kg) or high (10 IU/Kg) doses of intravenous insulin decreased more slowly and less in pregnant than in virgin rats. Feta! blood glucose levels were not affected by maternal insulin treatment. Results show that in the unanaesthesized late pregnant rat both insulin sensitivity and responsiveness decreased and it is proposed that this insulin resistance may represent a mechanism to delay disposal of ingested nutrients by maternal tissues, ensuring their availability to the fetus.

The in vivo ethanol elimination in human subjects, monkeys and rats was investigated after an oral ethanol dosage. After 0.4 g. ethanol/kg of body weight, ethanol elimination was much slower in human subjects than in monkeys. In order to detect a rise in monkey plasma ethanol concentrations as early as observed in human subjects, ethanol had to be administered at a dose of 3 g/kg body weight. Ethanol metabolism in rats was also much faster than in human subjects. However, human liver showed higher alcohol dchydrogenase activity and higher low Km aldehyde dchydrogenasc activity than rat liver. Thus, our data suggest a lack of relationship between hepatic ethanol-metabolizing activities and the in vivo ethanol elimination rate.

During the first half of gestation in the rat, maternal net body weight increases rapidly, whereas in the second half of gestation, the mass of maternal structures declines, coincident with the rate of maternal fat accumulation. Enhanced maternal food intake, extrahepatic tissue lipoprotein lipase (LPL) activity, and adipose tissue lipogenesis are responsible for the progressive accumulation of maternal fat. However, during late gestation, decreased fat synthesis in maternal adipose tissue, enhanced lipolytic activity, and decreased LPL activity deplete maternal fat depots. These changes, plus enhanced endogenous production of triglyceride-rich lipoproteins, are also responsible for maternal hypertriglyceridemia. This condition benefits the offspring in two ways: 1) enhanced LPL activity in maternal liver when fasting increases triglyceride consumption for ketone body synthesis, giving the basis for accelerated starvation; and 2) induction of LPL activity in the mammary gland before parturition diverts maternal circulating triglycerides to milk synthesis in preparation for lactation. The magnitude of the maternal-fetal glucose transfer was higher than that of any of the other substrates studied, including alanine, and despite actions to spare glucose, this transfer causes maternal hypoglycemia, which is especially intense in the fasting condition. This increases sympathoadrenal activity in the mother, which may contribute to her active gluconeogenesis. Glycerol was a more efficient glucose precursor than alanine and pyruvate, and whereas glycerol placental transfer is very small, it is proposed that the fetus benefits from this product of adipose tissue lipolysis when it is previously converted into glucose. In thyroidectomized pregnant rats treated with thyroxine for different periods, restraining maternal accumulation of fat depots during the early part of gestation compromises the normal metabolic adaptations during late gestation, including the capacity for accelerated starvation, which negatively affects fetal development.

I. Circulating [14C]glucose 2, 5 and 10 min after intravenous injection of [U- 14C]-L-alanine w~s greater in 24 hr starved than in fed rats. 2. In vitro uptake of [14C]alanine by liver and kidney cortex slices from 24 hr starved and fed rats rose in parallel with increased medium substrate concentration. 3. Formation of [14C]glucose from I mM (14C]alaninc was similar in liver and kidney cortex slices and increased in tissues from 24 hr starved compared with fed rat5. With 5 mM (14C]alanine more (14C]glucose was produced by liver than by kidney cortex slices from 24 hr starved rats. Liver ,lices always produced more [l4C]lactate and less [14C]-CO2 from [l 4C]alanine than kidney cortex slices. 4. It is proposed that under physiological conditions, the kidney~ cortex actively participates in glucose product10n from alanine.