The effects of different bill-trimming methods on the well-being of Pekin ducks.

Pekin ducks are often bill-trimmed to prevent feather pecking and cannibalism, but this practice has been criticized because of the resulting potential for acute and chronic pain. The goal of this experiment was to compare 2 different bill-trimming methods, hot blade trimming with cautery (TRIM) and cautery only (tip-searing; SEAR), on the behavior, bill morphology, and weight gain of Pekin ducks. Ducklings (n = 192, 96 per sex) were trimmed at the hatchery and assigned to 12 floor pens (3.66 x0.91 m) by treatment. Behavior was evaluated by scan sampling, and plumage condition was scored using a 0 to 3 scoring system. Thirty-six ducks were randomly euthanized at 3 and 6 wk of age, and their bills were collected for examination. Following fixation and decalcification, the bills were embedded in paraffin wax and sectioned longitudinally. Alternate sections were stained with hematoxylin and eosin and Masson's trichrome for the connective tissues, and with Bielschowsky's silver impregnation, Bodian's staining, and Holmes' staining for the nerve fibers. Trimmed ducks engaged in fewer bill-related behaviors and rested more than untrimmed ducks (NOTRIM) during the first 2 wk posttrim. Ducks in the SEAR and NOTRIM groups showed similar patterns of weight gain, but those in the TRIM group had a lower rate of gain than ducks in the SEAR group during the first week posttrim and had a lower rate of gain than those in the NOTRIM group for 2 wk posttrim. Feather scores of ducks in the NOTRIM group were significantly worse than those in the TRIM or SEAR group by 18 d, and scores continued to deteriorate at a greater rate than those of trimmed ducks throughout the study. Both trimming methods caused connective tissue proliferation in the bill stumps, but the TRIM method caused thicker scar tissue than the SEAR method. No neuromas were found with either trimming method, but there were more nerve fibers in bill stumps of the SEAR ducks than the TRIM ducks. These results suggest that acute pain is associated with both trimming methods, but that SEAR may be a preferable method, causing less check in weight gain and fewer bill morphological changes while still being effective in minimizing feather pecking damage.

Fatty acid and amino acid modulation of glucose cycling in isolated rat hepatocytes.

We studied the influence of glucose/glucose 6-phosphate cycling on glycogen deposition from glucose in fasted-rat hepatocytes using S4048 and CP320626, specific inhibitors of glucose-6-phosphate translocase and glycogen phosphorylase respectively. The effect of amino acids and oleate was also examined. The following observations were made: (1) with glucose alone, net glycogen production was low. Inhibition of glucose-6-phosphate translocase increased intracellular glucose 6-phosphate (3-fold), glycogen accumulation (5-fold) without change in active (dephosphorylated) glycogen synthase (GSa) activity, and lactate production (4-fold). With both glucose 6-phosphate translocase and glycogen phosphorylase inhibited, glycogen deposition increased 8-fold and approached reported in vivo rates of glycogen deposition during the fasted-->fed transition. Addition of a physiological mixture of amino acids in the presence of glucose increased glycogen accumulation (4-fold) through activation of GS and inhibition of glucose-6-phosphatase flux. Addition of oleate with glucose present decreased glycolytic flux and increased the flux through glucose 6-phosphatase with no change in glycogen deposition. With glucose 6-phosphate translocase inhibited by S4048, oleate increased intracellular glucose 6-phosphate (3-fold) and net glycogen production (1.5-fold), without a major change in GSa activity. It is concluded that glucose cycling in hepatocytes prevents the net accumulation of glycogen from glucose. Amino acids activate GS and inhibit flux through glucose-6-phosphatase, while oleate inhibits glycolysis and stimulates glucose-6-phosphatase flux. Variation in glucose 6-phosphate does not always result in activity changes of GSa. Activation of glucose 6-phosphatase flux by fatty acids may contribute to the increased hepatic glucose production as seen in Type 2 diabetes.

Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048.

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 microm). In livers of S4048-treated rats (0.5 mg kg(-1)min(-)); 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 +/- 2.0 versus 33.2 +/- 2.9 micromol kg(-1)min(-1)in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 +/- 1 versus 38 +/- 1%; p < 0.001) to glycogen synthesis (35 +/- 5% versus 65 +/- 5%; p < 0.005) by S4048. This was associated with a strong inhibition (-82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (approximately 9-fold), Glc-6-P translocase (approximately 4-fold), glycogen synthase (approximately 7-fold) and L-type pyruvate kinase (approximately 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis.

Measurement of the activation time of oxidative phosphorylation in isolated mouse hearts.

The purpose of this study was to develop a technique for determination of the dynamic regulation of oxidative myocardial metabolism in the mouse. The response time of myocardial oxygen consumption (MVO(2)) to a step in heart rate was determined in Langendorff-perfused mouse hearts. We examined the effect of glucose-only perfusate and glucose combined with 1, 3, or 6 mM pyruvate. Left ventricular systolic pressure (LVSP) decreased, yet the rate-pressure product (RPP) and MVO(2) increased with upward steps in heart rate. Pyruvate increased LVSP, RPP, and MVO(2) at the lower concentrations; however, when 6 mM pyruvate was added, LVSP and RPP became depressed while MVO(2) remained elevated. The mean response time of oxygen consumption to a step in heart rate from 270 to 350 beats/min was 9.8 s (n = 7) in the glucose-only perfused hearts. Perfusion with glucose plus 6 mM pyruvate decreased the response time to 5.3 s. These results are similar to those found in the rabbit heart and lay the groundwork for further examination of the dynamic regulation of oxidative myocardial metabolism in genetically altered mice. We concluded that the activation time of oxidative phosphorylation in the mouse is similar to that in larger species, despite the high mitochondrial content and natural heart rate of the mouse.

Increased hypoxic stress decreases AMP hydrolysis in rabbit heart.

OBJECTIVE: AMP conversion to adenosine by cytosolic 5'nucleotidase (5NT) or to IMP by AMP deaminase determines the degree of nucleotide degradation, and thus ATP resynthesis, during reoxygenation. To elucidate the regulation of AMP hydrolysis during ischemia, data from 31P NMR spectroscopy and biochemical analyses were integrated via a mathematical model. Since 5NT is downregulated during severe underperfusion (5% flow), we tested 5NT regulation during less severe underperfusion (10% flow) and then made the perfusate hypoxic to see if the greater stress reactivated 5NT. METHODS: 31P NMR spectra and coronary venous effluents were obtained from Langendorff-perfused rabbit hearts subjected to two 30-min periods of underperfusion (10% flow); the second period with or without additional hypoxia (30% O2). Data were analyzed with a mathematical model describing the kinetics of myocardial energetics and metabolism. RESULTS: A single 30-min period of 10% flow causes downregulation of AMP hydrolysis and the data from the second period of underperfusion are best described by lower 5NT activity, even in the presence of extra hypoxia. Thirty percent less purines appear in the venous effluent than predicted by the phosphoenergetics (PCr and ATP) when IMP is not allowed to accumulate by the model, however the model indicates that a constant accumulation of IMP via AMP deaminase could explain the discrepancy between expected and measured purines in the venous effluent. CONCLUSIONS: While AMP hydrolysis to adenosine is prominent in early ischemia and acts to preserve cellular energy potential, during a second ischemic period, nucleotides are conserved by the stable inhibition of AMP hydrolysis. Furthermore, during 10% flow conditions, nucleotides are conserved, possibly via an IMP-accumulatory pathway.

Downregulation of 5'-nucleotidase in rabbit heart during coronary underperfusion.

The hydrolysis of AMP to adenosine during acute coronary underperfusion is temporarily beneficial to myocardial survival yet may cause tissue injury during sustained underperfusion because of depletion of adenine nucleotides. We hypothesized that the enzyme mediating AMP hydrolysis, 5'-nucleotidase (5'-NT), is downregulated during sustained coronary underperfusion to prevent excessive loss of nucleotides. Langendorff-perfused rabbit hearts were subjected to two successive, identical 45-min periods of underperfusion (4-5% of baseline flow) separated by 20 min of reperfusion. Although coronary venous lactate efflux was comparable in the two periods, total coronary purine efflux during the second period of underperfusion was attenuated by 75%. Phosphorus nuclear magnetic resonance data showed that ATP fell 46% in the first period but fell only another 10% in the second period. Phosphocreatine levels fell comparably (75-78%) during both periods of underperfusion. Analysis using a mathematical model describing the kinetics of myocardial energetics revealed that the combined data set was best described by a lower activity of 5'-NT (52% decrease in maximal reaction velocity) during the second period of under-perfusion. Additional time course experiments showed that the decrease in 5'-NT activity was slow in onset, requiring approximately 20 min of underperfusion. The decrease in 5'-NT activity during sustained underperfusion may benefit tissue survival by limiting the depletion of myocardial adenine nucleotides. In conclusion, at the onset of coronary underperfusion, there is a high activity of 5'-NT, but later during sustained under-perfusion, 5'-NT is downregulated, resulting in decreased AMP hydrolysis to adenosine.

Open-system kinetics of myocardial phosphoenergetics during coronary underperfusion.

A novel hypothesis is proposed and tested describing open-system kinetics for myocardial phosphoenergetics. The hypothesis is that during severe coronary underperfusion there is precise matching of the rates of ATP synthesis and hydrolysis, but despite the precise balance of ATP rates, there is a decrease in the concentration of ATP and an increase in the concentration of phosphocreatine (PCr) caused by the hydrolysis of AMP to adenosine. Isolated rabbit hearts were perfused using a crystalloid medium, and coronary flow was reduced by 95% from baseline for 45 min followed by reperfusion. Phosphorus nuclear magnetic resonance spectroscopy showed a rapid decrease in PCr concentration to 25% of baseline at the onset of underperfusion followed by a gradual increase in PCr to 42% of baseline, while ATP decreased continuously to 65% of baseline. The kinetics of PCr and ATP could only be described by the precise matching of the rates of ATP synthesis and ATP hydrolysis and an open adenylate system that included the decrease in cytosolic AMP concentration via the production and efflux of adenosine. To confirm the hypothesis of open-system kinetics, two independent predictions were tested in separate experiments: 1) total coronary venous purine efflux (adenosine+inosine+hypoxanthine) during underperfusion was equal to the decrease in ATP concentration, and 2) there was no increase in PCr during moderate coronary underperfusion (80% flow reduction). In conclusion, the open nature of the myocardial adenylate system causes mass action effects that exert novel control over PCr and ATP concentrations during coronary underperfusion. The open-system kinetics cause ATP to decrease and PCr to increase, even though there is precise matching of the rates of ATP synthesis and hydrolysis. Finally, the hydrolysis of AMP to adenosine may benefit tissue survival during ischemia by improving the free energy of ATP hydrolysis, thereby delaying or preventing calcium overload.

Cell swelling and glycogen metabolism in hepatocytes from fasted rats.

Cell swelling is known to increase net glycogen production from glucose in hepatocytes from fasted rats by activating glycogen synthase. Since both active glycogen synthase and phosphorylase are present in hepatocytes, suppression of flux through phosphorylase may also contribute to the net increase in glycogen synthesis by cell swelling. We have developed an isotopic procedure to estimate the fluxes through glycogen synthase and phosphorylase in intact hepatocytes and we have examined the effect of cell swelling on both enzyme fluxes. The following observations were made. (1) Hypotonic or glutamine-induced cell swelling increased net glycogen production by activating flux through glycogen synthase with little effect on phosphorylase flux. Proline, previously shown to increase glycogen synthesis more than could be accounted for by its ability to cause cell swelling, increased flux through glycogen synthase and inhibited phosphorylase flux. (2) Incorporation of [14C]glucose into glycogen preceded complete mixing of [14C]glucose with the intracellular pool of UDPglucose. It is concluded that cell swelling affects glycogen synthase only and that UDPglucose is compartmentalized.

Carbamoyl phosphate and ureagenesis are not involved in amino-acid-stimulated glycogenesis.

Amino acids are known to stimulate glycogen synthesis via an increase in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959]. It has recently been postulated, however, that carbamoyl phosphate, an intermediate of ureagenesis, can function as a substrate for glucose phosphorylation via carbamoyl-phosphate:glucose phosphotransferase activity of the glucose-6-phosphatase system. This hypothesis would account for the stimulation of glycogenesis by amino acids such as glutamine and proline [Bode, A. M. & Nordlie, R. C. (1993) J. Biol. Chem. 268, 16298-16301]. To further examine the role carbamoyl phosphate may play in glycogenesis, isolated hepatocytes were incubated under a variety of conditions to manipulate ureagenesis, glycogenesis and carbamoyl-phosphate levels. Our data indicate that carbamoyl-phosphate levels do not correlate with amino-acid-stimulated glycogenesis and that ureagenesis and glycogenesis are not competing metabolic pathways.

Cell-swelling-induced taurine release from isolated perfused rat liver.

Astrocytes and lymphocytes are able to release significant amounts of taurine during periods of hypotonicity to reduce the increase in cell volume. To investigate this mechanism in the liver, we studied the release of free amino acids from isolated perfused rat liver during hypotonicity. The osmolarity of the perfusion medium was reduced from 305 to 255 or 205 mosM by decreasing the NaCl concentration 25 or 50 mM, respectively. This induced an 6-8% increase in liver mass and was associated with a specific 1.7-fold (-50 mosM) and 14-fold (-100 mosM) increase of the taurine release. None of the other amino acids measured showed a significant increase in their concentration in the effluent. The increase in taurine release occurred within 30 s after exposure to hypotonicity (maximal after 1-1.5 min) and followed closely the changes in liver mass. The taurine release declined gradually during successive exposures of the isolated liver to -100 mosM. This release was 29 and 17% of the original during the second and third exposure, respectively.

Cell swelling and the sensitivity of autophagic proteolysis to inhibition by amino acids in isolated rat hepatocytes.

In the isolated perfused rat liver, autophagic proteolysis is inhibited by hypo-osmotic perfusion media [Häussinger, D., Hallbrucker, C., vom Dahl, S., Lang, F. & Gerok, W. (1990) Biochem. J. 272, 239-242]. Here we report that in isolated hepatocytes, incubated in the absence of amino acids to ensure maximal proteolytic flux, proteolysis was not inhibited by hypo-osmolarity while the synthesis of glycogen from glucose, a process known to be very sensitive to changes in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959], was stimulated under identical conditions. However, in isolated hepatocytes, hypo-osmolarity increased the sensitivity of autophagic proteolysis to inhibition by low concentrations of amino acids. The anti-proteolytic effect of hypo-osmolarity in our experiments was not due to stimulation of amino-acid transport into the hepatocytes: neither the consumption of most amino acids, nor the rate of urea synthesis was appreciably affected by hypo-osmotic incubation conditions. In the course of these studies we also found that hypo-osmolarity increased the affinity of protein synthesis for amino acids. In the presence of amino acids the intracellular level of ATP was not much affected. However, because of cell swelling under these conditions the intracellular concentration of ATP decreased. It is proposed that a small part of the inhibition of proteolysis by amino acids may be due to this fall in ATP concentration.

Response during Sleep with Intervening Waking Amnesia.

During stage 1 sleep, subjects responded to suggestions on two or more nights, up to 5 months apart. While they were awake they did not recall the material to which they successfully responded while asleep on a subsequent night.