Effect of glucocorticoid treatment on glucose and glutamine metabolism by the small intestine of the rat

1. The effect of dexamethasone (30 micrograms day-1 100 g-1 body wt.) on the metabolism of glucose and glutamine was studied in the small intestine of rats after 9 days of treatment. 2. Dexamethasone treatment resulted in negative nitrogen balance (P less than 0.001), and produced increases in the concentrations of plasma glucose (22%, P less than 0.05), alanine (32%, P less than 0.001) and insulin (127%, P less than 0.001), but a decrease in the plasma concentration of glutamine (20%, P less than 0.05). 3. Portal-drained visceral blood flow increased by approximately 22% (P less than 0.001) in dexamethasone-treated rats, and was accompanied by a decrease in the arterio-venous concentration difference of glucose (43%, P less than 0.001) and an increase in that of lactate (22%, P less than 0.05), glutamine (35%, P less than 0.01), glutamate (33%, P less than 0.01) and alanine (21%, P less than 0.05). 4. Enterocytes isolated from dexamethasone-treated rats showed decreased and increased rates of glucose and glutamine utilization, respectively. 5. The maximal activities of hexokinase, 6-phosphofructokinase, citrate synthase and oxoglutarate dehydrogenase were decreased (30-64%, P less than 0.001) in intestinal mucosal scrapings of dexamethasone-treated rats, whereas the activity of glutaminase was increased (35%, P less than 0.001). 6. It is concluded that glucocorticoid administration decreases the rate of glucose utilization but increases that of glutamine (both in vivo and in vitro) by the epithelial cells of the small intestine. This may be caused by changes in the maximal activities of key enzymes in the pathways of glucose and glutamine metabolism in these cells.     

Glutamine and alanine metabolism in lungs of septic rats.

1. The metabolism of glutamine and alanine in the lung was studied in rats made septic by a caecal ligation and puncture technique. 2. The blood glucose concentration was not significantly different in septic rats, but blood pyruvate, lactate, glutamine and alanine concentrations were markedly increased as compared with sham-operated rats. Conversely, blood ketone body and plasma cholesterol concentrations were significantly decreased in septic rats. Both plasma insulin and plasma glucagon concentrations were markedly elevated in response to sepsis. Sepsis resulted in a negative nitrogen balance. 3. Sepsis increased the rates of production of glutamine (52.5%, P less than 0.001), alanine (38.9%, P less than 0.001) and glutamate (48.6%, P less than 0.001) by lung slices incubated in vitro. 4. Sepsis increased lung blood flow by 27.6% (P less than 0.05). Blood flow and arteriovenous concentration difference measurement across the lung of septic rats showed an increase in the net exchange rates of glutamine (142.5%, P less than 0.001), alanine (129.4%, P less than 0.001), glutamate (100.9%, P less than 0.001) and ammonia (138.0%, P less than 0.001) as compared with sham-operated control rats. 5. Sepsis produced significant decreases in the lung concentrations of glutamine (36.8%), glutamate (20.8%), 2-oxoglutarate (64.8%) and AMP (18.3%). The lung concentrations of alanine (95.9%), ammonia (67.7%) and pyruvate (89.7%) were increased. 6. The maximal activities of glutamine synthetase (20.4%, P less than 0.05), phosphate-dependent glutaminase (18.9%, P less than 0.05) and alanine aminotransferase (25.5%, P less than 0.05) were increased, but there was no marked change in that of glutamate dehydrogenase, in the lungs of septic rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skeletal muscle glutamine production in thermally injured rats

1. The effect of thermal injury (33-35% of body surface area) on the regulation of glutamine metabolism was studied in skeletal muscles of rats 7 days after injury. 2. Injury increased the rates of glutamine production in muscle, skin and adipose tissue preparations, with muscle production accounting for over 90% of total glutamine produced by the hindlimb. 3. Injury produced decreases in the concentrations of skeletal muscle glutamine (36%, P less than 0.001), glutamate (39%, P less than 0.001), alanine (24%, P less than 0.001), pyruvate (35%, P less than 0.001), 2-oxoglutarate (51%, P less than 0.001) and adenosine 5'-triphosphate (38%, P less than 0.001). The concentrations of ammonia (42%, P less than 0.001) and inosine 5'-phosphate (430%, P less than 0.001) were increased. 4. The maximal activity of glutamine synthetase was increased (22-40%, P less than 0.001) in muscles of injured rats, whereas that of glutaminase was unchanged. 5. Hindlimb blood flow decreased by approximately 15% in injured rats, which was accompanied by an enhanced net release of glutamine (80%, P less than 0.001) and alanine (44%, P less than 0.001). 6. It is concluded that there is an enhanced rate of release of both glutamine and alanine from skeletal muscle of thermally injured rats. This may be due to changes in efflux and/or increased intracellular formation of glutamine and alanine.     

Glucose and glutamine metabolism in the small intestine of septic rats

The intestinal metabolism of glucose and glutamine was studied in rats made septic by cecal ligation and puncture technique. Sepsis resulted in negative nitrogen balance and produced increases in the concentrations of blood pyruvate, lactate, alanine, and glutamine, and decreases in those of 3-hydroxybutyrate and acetoacetate. Both plasma insulin and glucagon concentrations were increased by 2.2- and 3.2-fold in septic rats, respectively. Portal-drained visceral blood flow increased in septic rats, and was accompanied by a decrease in the rates of utilization of glutamine and production of lactate, glutamate, and ammonia compared with those rates in sham-operated animals. Enterocytes isolated from septic rats showed decreased rates of glucose and glutamine utilization compared with cells isolated from corresponding controls. The maximal activities of hexokinase, 6-phosphofructokinase, pyruvate kinase, and glutaminase were decreased in intestinal mucosal scrapings of septic rats. It is concluded that a moderate form of sepsis decreases the rates of glucose and glutamine utilization (both in vivo and in vitro) by the epithelial cells of the small intestine. This may be caused by changes in the maximal activities of key enzymes in the pathways of glucose and glutamine metabolism in these cells as a metabolic adaptation to spare glucose and glutamine for use by other tissues.     

Hepatic glutamine metabolism in the septic rat.

1. The hepatic metabolism of glutamine, alanine, ammonia, urea, glutathione and glucose was studied in rats made septic by caecal ligation and puncture and was compared with that in rats that had undergone sham operation (laparotomy). 2. Sepsis resulted in increases in the plasma activities of gamma-glutamyltransferase (P less than 0.001), alanine aminotransferase (P less than 0.001) and aspartate aminotransferase (P less than 0.001), the serum total and direct bilirubin concentrations (P less than 0.001), and the blood lactate (P less than 0.01), glutamine (P less than 0.05), alanine (P less than 0.001) and urea (P less than 0.05) concentrations, but produced decreases in the blood ketone body (P less than 0.001) and glutathione (P less than 0.05) concentrations and in the plasma cholesterol concentration (P less than 0.05). These changes were associated with marked negative nitrogen balance in septic rats. 3. Sepsis increased total hepatic blood flow (by 22.7%) together with hepatic arterial flow (by 25.8%) and portal venous flow (by 18.7%). Sepsis resulted in marked increases in the net rates of hepatic extraction of glutamine (by 164%), alanine (by 138%) and ammonia (by 259%) with concomitant increases in the net rates of hepatic release of glutamate (by 105%), glutathione (by 87.5%), glucose (by 70.1%) and urea (by 100.4%). 4. Sepsis increased the activities of liver carbamoylphosphate synthase (by 16.4%), ornithine transcarbamylase (by 29.8%), argininosuccinate synthase (by 28.1%) and arginase (by 33.8%). 5. Septic rats exhibited marked increases in hepatic protein (by 46.0%), RNA (by 43.4%) and DNA (by 37.7%) contents. These changes were accompanied by marked increases in the activity of thymidine kinase (by 35.9%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hypothyroidism on glucose and glutamine metabolism by the gut of the rat.

1. The metabolism of glucose and glutamine was studied in the small intestine and the colon of rats after 4-5 weeks of hypothyroidism. 2. Hypothyroidism resulted in increases in the plasma concentrations of ketone bodies (P less than 0.05), cholesterol (P less than 0.001) and urea (P less than 0.001), but decreases in the plasma concentrations of free fatty acids (P less than 0.05) and triacylglycerol (P less than 0.001). These changes were associated with decreases in the plasma concentrations of total tri-iodothyronine, free tri-iodothyronine, total thyroxine and free thyroxine. 3. Hypothyroidism decreased both the DNA content (by 30.5%) and the protein content (by 23.6%) of intestinal mucosa, with the protein/DNA ratio remaining unchanged. The villi in the jejunum were shorter (P less than 0.05) and the crypt depth was decreased by about 26.5% in hypothyroid rats. 4. Portal-drained visceral blood flow showed no marked change in response to hypothyroidism, but was accompanied by decreased rates of extraction of glucose, lactate and glutamine and release of glutamate, alanine and ammonia. 5. Enterocytes and colonocytes isolated from hypothyroid rats showed decreased rates of utilization and metabolism of glucose and glutamine. 6. The maximal activities of hexokinase (EC 2.7.1.1), 6-phosphofructokinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), citrate synthase (EC 4.1.3.28), oxoglutarate dehydrogenase (EC 1.2.4.2) and phosphate-dependent glutaminase (EC 3.5.1.2) were decreased in intestinal mucosal scrapings from hypothyroid rats. Similar decreases were obtained in colonic mucosal scrapings (except for citrate synthase and oxoglutarate dehydrogenase) from hypothyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chronic uraemia on the formation of glucose and urea plus ammonia from L-alanine, L-glutamine and L-serine in isolated rat hepatocytes

The effects of chronic uraemia on glucose production and nitrogen release (urea plus ammonia formation) from alanine, glutamine or serine in isolated rat hepatocytes were studied. Uraemia increased the rate of formation of urea plus ammonia from all three amino acids by 38-93% when they were present at a final concentration of 10 mmol/l. At lower concentrations (2 mmol/l) the rate of nitrogen release was not significantly increased. Hepatocytes from normal rats whose food intake had been restricted to the level of that of uraemic rats did not show the increased rates of nitrogen release. The increased rates of nitrogen release with hepatocytes from uraemic rats were not accompanied by increased rates of glucose synthesis. Instead, accumulation of metabolic intermediates occurred: lactate and pyruvate (alanine or serine as substrates) and glutamate (glutamine as substrate). Livers of uraemic rats had increased activities of glutaminase (30%) and serine dehydratase (100%). Hepatocytes from normal rats treated with phlorhizin to increase the plasma glucagon/insulin ratio behaved in a similar manner to hepatocytes from uraemic rats. They had increased serine dehydratase activity, and increased rates of utilization of serine or glutamine. The possible implications of these findings for human uraemia are discussed.     

Maximal activity of phosphate-dependent glutaminase and glutamine metabolism in septic rats

The activity of phosphate-dependent glutaminase and glutamine metabolism by tissues known markedly to utilize or synthesize glutamine (or both) were studied in rats made septic by cecal ligation and puncture technique and compared with the same measures in rats that underwent sham operation (laparotomy). Blood glucose level was not markedly different in septic rats, but lactate, pyruvate, alanine, and glutamine levels were markedly increased. Conversely, blood ketone body concentrations were significantly decreased in septic rats. Both plasma insulin and glucagon levels were markedly elevated in response to sepsis. The maximal activity of phosphate-dependent glutaminase was decreased in the small intestine, increased in the kidney and mesenteric lymph nodes, and unchanged in the liver of septic rats. Arteriovenous concentration difference measurements across the gut showed a decrease in the net glutamine removed from the circulation in septic rats. Arteriovenous concentration difference measurements for glutamine showed that both renal uptake and skeletal muscle release of the amino acid were increased in response to sepsis, whereas measurements across the hepatic bed showed a net uptake of glutamine in septic rats. Enterocytes isolated from septic rats exhibited a decreased rate of utilization of glutamine and production of glutamate, alanine, and ammonia, whereas lymphocytes isolated from septic rats showed an enhanced rate of utilization of glutamine and production of glutamate, aspartate, and ammonia. It is concluded that, during sepsis, glutamine uptake and metabolism are enhanced in renal and lymphoid tissue but decreased in that of the small intestine, with increased rates of release by skeletal muscle; however, the liver appears to utilize glutamine in septic rats.     

Effects of Glutamine Deamination on Glutamine Deamidation in Rat Kidney Slices

Glutamate is known to inhibit the activity of isolated glutaminase I; however, its actual physiologic importance in regulating renal ammoniagenesis has not been established. To determine the regulatory role of glutamate on the metabolism of glutamine by rat kidney slices, we followed the effects on glutamine (2 mM) deamidation of increased removal of glutamate via augmented deamination. Three agents (malonate, 2,4-dinitrophenol, and methylene blue) were known to and shown here to hasten exogenous glutamate deamination. In slices from 10 control rats, 21.5+/-1.7 (SEM) mumol/g of ammonia were formed from amide nitrogen and 9.3+/-0.5 (SEM) mumol/g from the amino nitrogen of glutamine in vitro. Over 90% of the glutamine deamidated formed glutamate at one point in its catabolism. After addition of malonate (10 mM), 2,4-dinitrophenol (0.1 mM), or methylene blue (0.5 mM), the production of ammonia from the amino group rose to 29.3+/-6.0 (SEM) mumol/g, 20.0+/-1.8 (SEM) mumol/g, and 15.5+/-4.2 (SEM) mumol/g, respectively; ammonia production from the amide nitrogen rose also, 45.1+/-7.3 (SEM) mumol/g, 39.7+/-2.6 (SEM) mumol/g, and 41.9+/-3.7 (SEM) mumol/g. In the case of the former two, a minimum of 99% and 75% of the glutamine catabolized formed glutamate. Despite increased glutamine catabolism, there was no build up of glutamate in the media. A correlation between the formation of ammonia from the amino and amide nitrogen was apparent. Since none of the three agents selected affected phosphate activated glutaminase I activity directly or appeared to affect glutamine transport, we interpret the increase in deamidation as an expression of deinhibition of glutaminase I activity secondary to lowered glutamate concentrations at the deamidating sites through more rapid removal of glutamate via hastened deamination. Interestingly, slices removed from acidotic rats produced more ammonia from both the amino 29.1+/-3.8 (SEM) and amide nitrogens 45.9+/-4.3 (SEM) of glutamine, without a buildup of glutamate in the medium. At least 90% of the glutamine deamidated formed glutamate. A common mechanism is proposed to explain these results and the previous ones.     

Free amino acids in muscle tissue and plasma during exercise in man

Summary. Blood and muscle samples (percutaneous biopsy of m. quadriceps femoris) were taken before exercise and after 10 and 20 min of exercise in four healthy subjects working on a bicycle ergometer with a load of 70% of VO₂ max. Free amino acids were determined in plasma and muscle and the intracellular concentration of each amino acid was calculated by the chloride method. The plasma concentration of alanine, arginine and glutamine increased during exercise. In muscle there was an increase in glutamine and alanine concentration and a decrease in glutamate concentration at 10 min of exercise; after 20 min of exercise the increase in alanine and glutamine concentrations was less marked, but the decrease in glutamate concentration was of a similar magnitude. The results demonstrate that glutamate is a quantatively important ammonia acceptor during heavy exercise.     

Relation of renal cortical gluconeogenesis, glutamate content, and production of ammonia

Glutamate is an inhibitor of phosphate dependent glutaminase (PDG), and renal cortical glutamate is decreased in metabolic acidosis. It has been postulated previously that the rise in renal production of ammonia from glutamine in metabolic acidosis is due primarily to activation of cortical PDG as a consequence of the fall in glutamate. The decrease in cortical glutamate has been attributed to the increase in the capacity of cortex to convert glutamate to glucose in acidosis.In the present study, administration of ammonium chloride to rats in an amount inadequate to decrease cortical glutamate increased the capacity of cortex to produce ammonia from glutamine in vitro and increased cortical PDG. Similarly, cortex from potassium-depleted rats had an increased capacity to produce ammonia and an increase in PDG, but glutamate content was normal. The glutamate content of cortical slices incubated at pH 7.1 was decreased, and that at 7.7 was increased, compared to slices incubated at 7.4, yet ammonia production was the same at all three pH levels. These observations suggest that cortical glutamate concentration is not the major determinant of ammonia production.In potassium-depleted rats there was a 90% increase in the capacity of cortex to convert glutamate to glucose, yet cortical glutamate was not decreased. In vitro, calcium more than doubled conversion of glutamate to glucose by cortical slices without affecting the glutamate content of the slices, and theophylline suppressed conversion of glutamate to glucose yet decreased glutamate content. These observations indicate that the rate of cortical gluconeogenesis is not the sole determinant of cortical glutamate concentration.The increase in cortical gluconeogenesis in acidosis and potassium depletion probably is not the primary cause of the increase in ammonia production in these states, but the rise in gluconeogenesis may contribute importantly to the maintenance of increased ammoniagenesis by accelerating removal of the products of glutamine degradation.     

Effects of hypernatremia on organic brain osmoles.

We studied the effects of varying degrees and durations of hypernatremia on the brain concentrations of organic compounds believed to be important, so-called "idiogenic" osmoles in rats by means of conventional biochemical assays, nuclear magnetic resonance spectroscopy, and high-performance liquid chromatography. There were no changes in the concentrations of these osmoles (specifically myoinositol, sorbitol, betaine, glycerophosphorylcholine [GPC], phosphocreatine, glutamine, glutamate, and taurine) in rats with acute (2 h) hypernatremia (serum Na 194 +/- 5 meq/liter). With severe (serum Na 180 +/- 4 meq/liter) chronic (7 d) hypernatremia, the concentrations of each of these osmoles except sorbitol increased significantly: myoinositol (65%), betaine (54%), GPC (132%), phosphocreatine (73%), glutamine (143%), glutamate (84%), taurine (78%), and urea (191%). Together, these changes account for 35% of the change in total brain osmolality. With moderate (serum Na 159 +/- 3 meq/liter) hypernatremia, more modest but significant increases in the concentrations of each of these osmoles except betaine and sorbitol were noted. When rats with severe chronic hypernatremia were allowed to drink water freely, their serum sodium as well as the brain concentrations of all of these organic osmoles except myoinositol returned to normal within 2 d. It is concluded that: idiogenic osmoles play an important role in osmoregulation in the brain of rats subjected to hypernatremia; the development of these substances occur more slowly than changes in serum sodium; and the decrease in concentration of myoinositol occurs significantly more slowly than the decrease in serum sodium which occurs when animals are allowed free access to water. These observations may be relevant to the clinical management of patients with hypernatremia.     

Effects of Parathyroid Hormone on Skeletal Muscle Protein and Amino Acid Metabolism in the Rat

Because prominent skeletal muscle dysfunction and muscle wasting are seen in both chronic uremia and in primary hyperparathyroidism, and because markedly elevated parathyroid hormone levels occur in both disorders, potential effects of parathyroid hormone on skeletal muscle protein, amino acid, and cyclic nucleotide metabolism were studied in vitro using isolated intact rat epitrochlearis skeletal muscle preparations. Intact bovine parathyroid hormone and the synthetic 1-34 fragment of this hormone stimulated the release of alanine and glutamine from muscle of control but not from chronically uremic animals. This stimulation was dependent upon the concentration of parathyroid hormone added: At 10⁵ ng/ml parathyroid hormone increased alanine release 84% and glutamine release 75%. Intracellular levels of alanine and glutamine were not altered by parathyroid hormone. Increasing concentrations of the 1-34 polypeptide decreased [³H]leucine incorporation into protein of muscles from both control and uremic animals. Using muscles from animals given a pulse-chase label of [guanido-¹⁴C]arginine in vivo, parathyroid hormone increased the rate of loss of ¹⁴C label from acid-precipitable protein during incubation and correspondingly increased the rate of appearance of this label in the incubation media. Parathyroid hormone increased muscle cAMP levels by 140% and cGMP levels by 185%, but had no effect on skeletal muscle cyclic nucleotide phosphodiesterase activities as assayed in vitro. Adenylyl cyclase activity in membrane preparations from control but not uremic rats was stimulated by parathyroid hormone in a concentration-dependent fashion. However, no stimulation of guanylyl cyclase activity was noted by parathyroid hormone, although stimulation by sodium azide was present. Incubation of muscles with added parathyroid hormone produced a diminished responsiveness towards epinephrine or serotonin regulation of amino acid release and cAMP formation in the presence compared to the absence of parathyroid hormone. In the absence of parathyroid hormone, detectable inhibition of alanine and glutamine release was produced by 10⁻⁹ M epinephrine, whereas in the presence of parathyroid hormone (1,000 ng/ml) inhibition of alanine and glutamine release required 10⁻⁶ M or greater epinephrine. Resistance to cyclic AMP action as well as inhibition of cyclic AMP formation by parathyroid hormone was found. Preincubation of rat sarcolemma with 1-34 parathyroid hormone produced a decreased activity of the isoproterenol-stimulable adenylyl cyclase activity but there was no apparent change in the concentration of isoproterenol required for one-half maximal and maximal stimulation of the enzyme.     

Oral glutamine attenuates indomethacin-induced small intestinal damage

The use of NSAIDs (non-steroidal anti-inflammatory drugs), although of great therapeutic value clinically, is limited by their tendency to cause mucosal damage in the gastrointestinal tract. In the small intestine, the effects these drugs have been shown to produce include inhibition of cyclo-oxygenase, mitochondrial dysfunction and free radical-induced oxidative changes, all of which contribute to the mucosal damage seen. Glutamine is a fuel preferentially used by enterocytes and is known to contribute to maintaining the integrity of these cells. In the present study, we investigated the effect of glutamine on indomethacin-induced changes in the small intestinal mucosa. Rats were given 2% glutamine or glutamic acid or isonitrogenous amino acids, glycine or alanine, in the diet for 7 days. Indomethacin was then administered orally at a dose of 40 mg/kg of body weight. After 1 h, the small intestine was removed and used for the measurement of parameters of oxidative stress and mitochondrial and BBM (brush border membrane) function. Evidence of oxidative stress was found in the mucosa of the small intestine of drug-treated rats, as indicated by significantly increased activity of xanthine oxidase (P < 0.001) and myeloperoxidase (P < 0.001), with corresponding decreases in the levels of several free radical scavenging enzymes and alpha-tocopherol (P < 0.001 in all cases). Levels of products of peroxidation were also significantly elevated (P < 0.001 for all the parameters measured). In addition, oxidative stress was evident in isolated intestinal mitochondria and BBMs (P < 0.001 for all the parameters measured), with associated alterations in function of these organelles (P < 0.001 for all the parameters measured). Supplementation of the diet with glutamine or glutamic acid prior to treatment with indomethacin produced significant amelioration in all the effects produced by the drug in the small intestine (P < 0.001 for all the parameters measured). Glycine and alanine were found to be much less effective in these respects.     

Hepatobiliary disposition of a drug/metabolite pair: Comprehensive pharmacokinetic modeling in sandwich-cultured rat hepatocytes

The hepatobiliary disposition of xenobiotics may involve passive and/or active uptake, metabolism by cytochromes P450, and excretion of the parent compound and/or metabolite(s) into bile. Although in vitro systems have been used to evaluate these individual processes discretely, mechanistic in vitro studies of the sequential processes of uptake, metabolism, and biliary or basolateral excretion are limited. The current studies used sandwich-cultured (SC) rat hepatocytes combined with a comprehensive pharmacokinetic modeling approach to investigate the hepatobiliary disposition of terfenadine and fexofenadine, a model drug/metabolite pair. The metabolism of terfenadine and the biliary excretion of terfenadine and fexofenadine were determined in control and dexamethasone-treated SC rat hepatocytes. Dexamethasone (DEX) treatment increased the formation rates of the terfenadine metabolites azacyclonol and fexofenadine approximately 20- and 2-fold, respectively. The biliary excretion index (BEI) of fexofenadine, when generated by terfenadine metabolism, was not significantly different from the BEI of preformed fexofenadine (15 +/- 2% versus 19 +/- 2%, respectively). Pharmacokinetic modeling revealed that the rate constant for hepatocyte uptake was faster for terfenadine compared with preformed fexofenadine (2.5 versus 0.08 h(-1), respectively), whereas the biliary excretion rate constant for preformed fexofenadine exceeded that of terfenadine (0.44 versus 0.039 h(-1), respectively). Interestingly, the rate constants for basolateral excretion of terfenadine and fexofenadine were comparable (3.2 versus 1.9 h(-1), respectively) and increased only slightly with DEX treatment. These studies demonstrate the utility of the SC hepatocyte model, coupled with pharmacokinetic modeling, to evaluate the hepatobiliary disposition of generated metabolites.     

Hypothermia induces interleukin-10 and attenuates injury in the lungs of endotoxemic rats

We recently reported that hypothermia protects against intrapulmonary nitric oxide overproduction and nitric oxide-mediated lung injury in endotoxemic rats. Few studies have been performed to investigate whether hypothermia reduces inflammation by affecting favorable changes in chemokine and pro- and anti-inflammatory cytokine profiles. In this study, we tested the hypothesis that hypothermia decreases concentrations of growth-related oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1), interleukin (IL)-1beta, IL-6, and myeloperoxidase and increases concentration of IL-10 in the lungs endotoxemic rats. Twelve rats were anesthetized and randomized to treatment with either hypothermia (T = 18-24 degrees C; n = 6) or normothermia (T = 36-38 degrees C, n = 6). Endotoxin (15 mg/kg of Escherichia coli lipopolysaccharide) was administered intravascularly and lung tissue was harvested 150 min later. Three additional rats were sham instrumented and maintained as normothermic but not given endotoxin. Hematoxylin & eosin staining was performed for qualitative inspection of tissues. Quantitative analyses of lung homogenates were performed using enzyme-linked immunosorbent assays for IL-1beta, IL-6, IL-10, and GRO/CINC-1. Myeloperoxidase concentrations were determined using a colorimetric assay. Hypothermia attenuated the induction of intrapulmonary IL-1beta (P < 0.05), IL-6 (P < 0.05), GRO/CINC-1 (P < 0.05), and myeloperoxidase (P < 0.05) caused by endotoxin. Inspection of the lungs revealed that hypothermia similarly attenuated histological signs of injury, such as interstitial edema and neutrophil accumulation. Hypothermia increased the intrapulmonary concentration of IL-10 more than 3-fold over that measured in the normothermia (endotoxin-exposed) group (P < 0.05). Hypothermia inhibits neutrophil recruitment in the lungs of endotoxemic rats in part by decreasing proinflammatory cytokine expression. Additionally, hypothermia induces intrapulmonary IL-10 expression. Further studies are needed to investigate whether IL-10 mediates the anti-inflammatory effects of hypothermia.     

Effects in vivo of decreased plasma and intracellular muscle glutamine concentration on whole-body and hindquarter protein kinetics in rats.

Glutamine is considered to be a 'conditionally' essential amino acid. During situations of severe stress like sepsis or after trauma there is a fall in plasma glutamine levels, enhanced glutamine turnover and intracellular muscle glutamine depletion. Under these conditions, decreased intramuscular glutamine concentration correlates with reduced rates of protein synthesis. It has therefore been hypothesized that intracellular muscle glutamine levels have a regulatory role in muscle protein turnover rates. Administration of the glutamine synthetase inhibitor methionine sulphoximine (MSO) was used to decrease glutamine levels in male Wistar rats. Immediately after the MSO treatment (t=0 h), and at t=6 h and t=12 h, rats received intraperitoneal injections (10 ml/100 g body weight) with glutamine (200 mM) to test whether this attenuated the fall in plasma and intracellular muscle glutamine. Control animals received alanine and saline after MSO treatment, while saline was also given to a group of normal rats. At t=18 h rats received a primed constant infusion of L-[2,6-3H]phenylalanine. A three-pool compartment tracer model was used to measure whole-body protein turnover and muscle protein kinetics. Administration of MSO resulted in a 40% decrease in plasma glutamine and a 60% decrease in intracellular muscle glutamine, both of which were successfully attenuated by glutamine infusions. The decreased intracellular muscle glutamine levels had no effect on whole-body protein turnover or muscle protein kinetics. Also, glutamine supplementation did not alter these parameters. Alanine supplementation increased both hindquarter protein synthesis and breakdown but the net balance of phenylalanine remained unchanged. In conclusion, our results show that decreased plasma and muscle glutamine levels have no effect on whole-body protein turnover or muscle protein kinetics. Therefore, it is unlikely that, in vivo, the intracellular muscle concentration of glutamine is a major regulating factor in muscle protein kinetics.     

The effect of glucocorticoids on thyrotropin secretion

The effect of large doses of glucocorticoids on thyrotropin (TSH) secretion in normal and hypothyroid humans has been studied. Plasma TSH concentrations were measured before, during, and after treatment with dexamethasone given orally for 24-48 hr. In 17 patients with primary hypothyroidism, plasma TSH levels fell significantly during treatment to a mean of 54% of control (range 23-96%). Within 48 hr after the withdrawal of dexamethasone, TSH concentrations transiently increased above pretreatment values. The mean increase was to 156% of control (range 106-294). Similar changes, but of smaller magnitude, were observed in 15 normal subjects. Administration of single oral doses of dexamethasone and oral or intravenous doses of cortisol were followed by reduction of plasma TSH levels to 18-47% of control within 8-12 hr in eight hypothyroid patients. This fall also was followed by significant TSH rises above control values before they returned to the pretreatment levels. Mineralocorticoid administration was not followed by any changes in plasma TSH concentrations in three subjects.TSH responses to steroid were also studied in rats. In hypothyroid rats given dexamethasone intravenously, plasma TSH fell to 63% of control in 30-90 min and then returned to normal or above in 3-4 hr. Dexamethasone also reduced plasma TSH concentrations in normal rats but no rebound was observed in these animals. Dexamethasone did not block the increase in plasma TSH produced by thyrotropin releasing factor (TRF) administration in vivo. Neither basal nor TRF-mediated TSH release from hemipituitaries in vitro was reduced by dexamethasone or corticosterone. These studies indicate that glucocorticoids reduce TSH secretion and suggest that this effect occurs at a suprahypophyseal level.     

Ammonia production by individual segments of the rat nephron

Ammonia production was measured directly in 10 segments of the rat nephron to determine the relative importance of the segments as sites of renal ammonia production. Tubules were microdissected from normal rats and rats drinking 0.28 M NH4Cl or 0.28 M NaHCO3 for 3-8 d. The segments were incubated in vitro with and without 2 mM glutamine. Ammonia concentrations in the incubation fluid were measured by microfluorometry to determine ammonia production rates. All segments produced ammonia from glutamine. In normal rats, production with glutamine was highest (greater than 5 pmol/min per mm) in the proximal convoluted (S-1), proximal straight (S-3), and distal convoluted tubules, and lowest (less than or equal to 2) in cortical and medullary collecting ducts and thin descending limbs. Metabolic acidosis increased production by 60% in the S-1 segment of the proximal convoluted tubule and by 150% in the S-2 segment of the proximal straight tubule without significant effect in any other segment. Bicarbonate loading decreased production by S-1 but had no effect on S-2 or S-3. Thus, acid-base changes altered production only in specific segments of the proximal tubule. We infer that the bulk of ammonia production occurs in the proximal tubules and that production by collecting ducts can account for only a few percent of renal ammonia production and excretion in the rat.     

Positive end-expiratory pressure modulates local and systemic inflammatory responses in a sepsis-induced lung injury model

OBJECTIVE: Previous animal studies have shown that certain modes of mechanical ventilation (MV) can injure the lungs. Most of those studies were performed with models that differ from clinical causes of respiratory failure. We examined the effects of positive end-expiratory pressure (PEEP) in the setting of a clinically relevant, in vivo animal model of sepsis-induced acute lung injury ventilated with low or injurious tidal volume. METHODS: Septic male Sprague-Dawley rats were anesthetized and randomized to spontaneous breathing or four different strategies of MV for 3 h at low (6 ml/kg) or high (20 ml/kg) tidal volume (V(T)) with zero PEEP or PEEP above inflection point in the pressure-volume curve. Sepsis was induced by cecal ligation and perforation. Mortality rates, pathological evaluation, lung tissue cytokine gene expression, and plasma cytokine concentrations were analyzed in all experimental groups. RESULTS: Lung damage, cytokine synthesis and release, and mortality rates were significantly affected by the method of MV in the presence of sepsis. PEEP above the inflection point significantly attenuated lung damage and decreased mortality during 3 h of ventilation with low V(T) (25% vs. 0%) and increased lung damage and mortality in the high V(T) group (19% vs. 50%). PEEP attenuated lung cytokine gene expression and plasma concentrations during mechanical ventilation with low V(T). CONCLUSIONS: The use of a PEEP level above the inflection point in a sepsis-induced acute lung injury animal model modulates the pulmonary and systemic inflammatory responses associated with sepsis and decreases mortality during 3 h of MV.