Krebs-Henseleit Bicarbonate Research Articles

295 Synthesis of glycine from trans-4-hydroxy-L-proline in tissues of juvenile hybrid striped bass (Morone chrysops x M. Saxatilis)

Abstract Trans-4-Hydroxy-L-Proline (Hyp) is a major amino acid in the collagen of fish. The turnover of collagen in various tissues releases a large amount of Hyp, which has traditionally been considered as a metabolic waste. However, results of recent studies have identified the conversion of Hyp into glycine in piglets. The present study tests this hypothesis that hybrid striped bass (HSB, a carnivorous fish species) can form glycine from Hyp. Fish were fed a standard diet containing 60% fishmeal for 10 wk and grew from 5.5 to 45 g during a 9-wk period. Slices (~75 mg) of skeletal muscle, liver, proximal intestine, gills, and brain were obtained from 45-g HSB (n = 6) and incubated at 26 °C for 2 h in 1 mL of oxygenated (95% O2/5% CO2) Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5 mM D-glucose) containing 0, 0.2, or 2 mM Hyp. After a period of 2 h incubation, 200 μL of 1.5 M HClO4 was added into the incubation medium to stop the reaction. The acidified medium plus tissue together was homogenized and the homogenizer was rinsed with 1 mL of water, followed by the addition of 100 μL of 2 M K2CO3. The neutralized extract was analyzed for amino acids using HPLC involving o-phthaldialdehyde derivatization. Tissues without incubation were also determined for amino acids. Data were statistically analyzed by a paired t-test. Glycine was produced from 0.2 and 2 mM Hyp by the liver and skeletal muscle, and 2 mM Hyp by the intestine (Table 1). Glycine was further converted into serine in the liver, but not skeletal muscle or intestine. The production of glycine or serine from Hyp was not due to net protein breakdown in tissues. Tyrosine was measured as an indicator of protein degradation. There was no detectable generation of glycine from 0.2 or 2 mM Hyp by the gills or brain of HSB. These results indicate the synthesis of glycine from Hyp by juvenile HSB in a tissue-specific manner. Because skeletal muscle accounts for ~45% of the body weight, this organ is the major site for this metabolic pathway in the fish. (Supported by USDA/NIFA grant No. 2022-67015-36200)

384 Synthesis of glycine from trans-4-hydroxy-L-proline by placentae but not endometria of gestating gilts

Abstract Trans-4-hydroxy-L-proline (Hyp) is an abundant amino acid in the collagen of pigs. The turnover of this protein releases a large amount of Hyp, traditionally considered a metabolic waste. However, results of recent studies documented the conversion of Hyp into glycine in young pigs. Because pregnant gilts are typically fed a soybean meal-based diet that contains insufficient glycine and provides only 35% of the total glycine needed by pregnant gilts, it is possible that the conceptus can form glycine from Hyp. This hypothesis was tested in the present study. Gilts were fed a 1.8-kg corn- and soybean meal-based diet containing 14% crude protein. On d 90 of gestation, gilts (n = 5) were hysterectomized after euthanasia. Slices (~ 100 mg) of placentae or endometria were incubated at 37 °C for 2 h in oxygenated (95% O2/5% CO2) Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5 mM D-glucose) containing 0 or 2 mM Hyp. After a period of 2 h incubation, 200 μL of 1.5 M HClO4 was added into the incubation medium to stop the reaction. The tissues were homogenized and rinsed with 1 mL water, followed by the addition of 100 μL of 2 M K2CO3. The neutralized extract was analyzed for amino acids using HPLC involving derivatization with o-phthaldialdehyde. Tissues that received no incubation time were determined as well. Data were analyzed by the paired t-test. Results indicated that glycine was produced from 2 mM Hyp by the placenta (Table 1). Glycine was subsequently converted into serine by the placenta. The formation of glycine and serine from Hyp was not due to protein breakdown because there were no differences (P > 0.05) in concentrations of tyrosine (an amino acid that is neither synthesized nor degraded by porcine placentae) in the incubation medium plus tissue at the end of a 2-h incubation period. In contrast to placentae, endometria did not form glycine or serine from Hyp. These findings indicate an important role for Hyp in the provision of both glycine and serine in the conceptus, which may be nutritionally and physiologically significant to compensate for a deficiency of glycine in maternal diets for gestating gilts. (Supported by a USDA/NIFA grant # 2022-67015-36376 and USDA multistate Hatch project #7752: S1081)

Effect of esaxerenone on ischemia and reperfusion injury in rat hearts.

In myocardial infarction, the addition of mineralocorticoid receptor blockers (MRBs) to standard therapies, such as angiotensin-converting enzyme inhibitors or beta-blockers, reportedly reduces mortality and cardiac events. We investigated whether the non-steroidal MRB esaxerenone has cardioprotective effects and its protective mechanisms. Isolated rat hearts were Langendorff-perfused (constant pressure, 80 mmHg) with oxygenated Krebs-Henseleit bicarbonate buffer and reperfused for 60 min; afterwards, recovery of function (left ventricular pressure, measured with an intraventricular balloon) and myocardial injury were measured. In a preliminary study, we determined the optimal concentration of esaxerenone required for myocardial protection. Next, esaxerenone was administered in the pre- and post-ischemic phases to determine the optimal timing of administration. In addition, we assessed coronary flow response to acetylcholine (ACh) with and without esaxerenone. We examined whether esaxerenone-induced cardioprotection was prevented by targeting putative components in the preconditioning manner (the mitochondrial ATP-sensitive potassium (KATP) channel). Myocardial protection by esaxerenone was observed when esaxerenone was administered before ischemia but not after ischemia. The coronary flow response to ACh was significantly better in the esaxerenone group than in the control group. The cardioprotective effect of esaxerenone was eliminated by the mitochondrial KATP channel blocker, 5-hydroxy decanoate. This study confirmed the myocardial protective effect of the pre-ischemic administration of esaxerenone. Esaxerenone may contribute to coronary endothelial protection and exert pharmacological preconditioning via the mitochondrial KATP channel.

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model.

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Oxidation of amino acids, glucose, and fatty acids as metabolic fuels in enterocytes of developing pigs.

Enterocytes of young pigs are known to use glutamine, glutamate, and glucose as major metabolic fuels. However, little is known about the roles of aspartate, alanine, and fatty acids as energy sources for these cells. Therefore, this study simultaneously determined the oxidation of the amino acids and glucose as well as short- and long-chainfatty acids in enterocytes of developing pigs. Jejunal enterocytes were isolated from 0-, 7-, 14- and 21-day-old piglets, and incubated at 37°C for 30min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5mM D-glucose and one of the following: D-[U-14C]glucose, 0.5-5mM L-[U-14C]glutamate, 0.5-5mM L-[U-14C]glutamine, 0.5-5mM L-[U-14C]aspartate, 0.5-5mM L-[U-14C]alanine, 0.5-2mM L-[U-14C]palmitate, 0.5-5mM [U-14C]propionate, and 0.5-5mM [1-14C]butyrate. At the end of the incubation, 14CO2 produced from each 14C-labeled substrate was collected. Rates of oxidation of each substrate by enterocytes from all age groups of piglets increased (P < 0.05) gradually with increasing its extracellular concentrations. The rates of oxidation of glutamate, glutamine, aspartate, and glucose by enterocytes from 0- to 21-day-old pigs and of alanine from newborn pigs were much greater (P < 0.05) than those for the same concentrations of palmitate, propionate, and butyrate. Compared with 0-day-old pigs, the rates of oxidation of glutamate, aspartate, glutamine, alanine, and glucose by enterocytes from 21-day-old pigs decreased (P < 0.05) markedly, without changes in palmitate oxidation. Oxidation of alanine, propionate, butyrate and palmitate by enterocytes of pigs was limited during their postnatal growth. At each postnatal age, the oxidation of glutamate, glutamine, aspartate, and glucose produced much more ATP than alanine, propionate, butyrate and palmitate. The degradation of glutamate was initiated primarily by glutamate-pyruvate and glutamate-oxaloacetatetransaminases. Our results indicated that amino acids (glutamate plus glutamine plus aspartate) are the major metabolic fuels in enterocytes of 0- to 21-day-old pigs.

Oxidation of amino acids, glucose, and fatty acids as metabolic fuels in enterocytes of post-hatching developing chickens.

This study determined the oxidation of amino acids, glucose and fatty acid in enterocytes of developing chickens. Jejunal enterocytes were isolated from 0-, 7-, 21-, and 42-d-old broiler chickens, and incubated at 40°C for 30min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5mM D-glucose and one of the following: 0.5-5mM L-[U-14C]glutamate, 0.5-5mM L-[U-14C]glutamine, 0.5-5mM L-[U-14C]aspartate, 0.5-5mM L-[U-14C]alanine, 0.5-2mM [U-14C]palmitate, D-[U-14C]glucose, 0.5-5mM [U-14C]propionate, and 0.5-5mM [1-14C]butyrate. 14CO2 produced from each 14C-labeled substrate was collected for determination of radioactivity. Among all the substrates studied, glutamate had the greatest rate of oxidation in enterocytes from 0- to 42-d-old chickens. Glutamate transaminases, rather than glutamate dehydrogenase, may be primarily responsible for initiating glutamate degradation. Rates of amino acid and fatty acid oxidation by cells increased (P < 0.05) with increasing their extracellular concentrations from 0.5 to 5mM. Rates of glutamate and glucose oxidation in enterocytes decreased (P < 0.05) with increasing age, and rates of glutamine, aspartate, propionate, and butyrate oxidation were lower (P < 0.05) in 42-d-old chickens than in 0-d-old chickens. By contrast, oxidation of palmitate at 2mM increased (P < 0.05) by 118% in cells from 42-d-old chickens, compared with 0-d-old chickens. Compared with glutamate, oxidation of glutamine, aspartate, alanine, propionate, butyrate, and palmitate was limited in cells from all age groups of chickens. Collectively, these results indicate that glutamate is the major metabolic fuel in enterocytes of 0- to 42-d-old chickens.

Oxidation of energy substrates in tissues of largemouth bass (Micropterus salmoides).

This study tested the hypothesis that amino acids are oxidized at higher rates than glucose and palmitate for ATP production in tissues of largemouth bass (LMB, a carnivorous fish). Slices (10 to 50 mg) of liver, proximal intestine, kidney, and skeletal muscle isolated from LMB were incubated at 26°C for 2h in oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5mM D-glucose) containing either D-[U-14C]glucose, 2mM L-alanine plus L-[U-14C]alanine, 2mM L-aspartate plus L-[U-14C]aspartate, 2mM L-glutamate plus L-[U-14C]glutamate, 2mM L-glutamine plus L-[U-14C]glutamine, 2mM L-leucine plus L-[U-14C]leucine, or 2mM palmitate plus [U-14C]palmitate. In parallel experiments, tissues were incubated with a [U-14C]-labeled tracer and a mixture of unlabeled substrates [alanine, aspartate, glutamate, glutamine, leucine, and palmitate (2mM each) plus 5mM glucose]. 14CO2 was collected to calculate the rates of substrate oxidation. In separate experiments, O2 consumption by each tissue was measured in the presence of individual or a mixture of substrates. The activities of key metabolic enzymes were also measured. Results indicated that the liver and skeletal muscle had a limited ability to oxidize glucose and palmitate to CO2 for ATP production in the presence of individual or a mixture of substrates due to low activities of carnitine palmitoyltransferase-I, hexokinase and pyruvate dehydrogenase. In the presence of individual substrates, each amino acid was actively oxidized by all the tissues. In the presence of a mixture of substrates, glutamine and glutamate were the major metabolic fuels in the proximal intestine and kidney, as glutamine for the liver and aspartate for skeletal muscle. All the tissues had high activities of glutaminase, glutamate dehydrogenase, and transaminases. At the same extracellular concentration of amino acids (2 mM) in a mixture of energy substrates, glutamine was the major metabolic fuel for the liver of the LMB, glutamine and glutamate for the proximal intestine and kidneys, and aspartate for the skeletal muscle. Glutamine plus glutamate plus aspartate generated 60-70% of ATP in LMB tissues.

220-LB: Glucagon Promotes Hepatic Autophagy by AMPK-Mediated mTORC1 Inhibition

Glucagon plays an important role in hepatic glucose metabolism as well as protein/amino acid metabolism but detailed knowledge about glucagon’s role in hepatic autophagy is limited. We hypothesized that glucagon regulate hepatic protein metabolism partly through activation of AMP-kinase (AMPK) that in turn lead to inhibition of mammalian target of rapamycin complex1 (mTORC1) and activation of Uncoordinated-51 like autophagy activating kinase-1 (ULK1) resulting in increased autophagy. In order to test this hypothesis, we infused chronically catheterized awake mice with [13C5]-glutamine [0.225 mg/(kg-min)] for 120 min after which a second line was connected infusing somatostatin [4 µg/(kg-min)] and insulin [0.1 mU/(kg-min)], with (n=7) or without glucagon [10 ng/(kg-min)] (n=6) for 90 min. This raised plasma glucagon concentration ~18-fold compared to control. Glucagon infusion increased phosphorylation of hepatic: AMPKThr172, RaptorSer792, ULK1Ser555 (P=0.03, P=0.03, and P=0.06, respectively, compared to control), and resulted in hepatic Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II accumulation (P=0.02), a marker of autophagy, assessed by immunoblotting. Glucagon treatment also increased the 13C enrichment (m+3) in liver glucose (P&amp;lt;0.0001), indicating increased conversion of [13C5]-glutamine to glucose as well as increased plasma urea concentrations (P=0.04). Consistent with these in vivo results, parallel studies performed in liver slices obtained from overnight fasted male rats (n=6) incubated in Krebs-Henseleit bicarbonate buffer supplemented with [3-13C]-alanine (1 mM), glucagon (250 µM) treatment increased: glucose production (P=0.01), urea production (P=0.0005), and intracellular levels of leucine (P=0.045). In conclusion, our data suggest that glucagon promotes hepatic proteolysis and ureagenesis by AMPK-mediated suppression of mTOC1 activity, leading to increased hepatic autophagy. Disclosure K.D. Galsgaard: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp &amp; Dohme Corp. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc. Advisory Panel; Spouse/Partner; Merck &amp; Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. K. Petersen: Advisory Panel; Spouse/Partner; Merck Sharp &amp; Dohme Corp. Research Support; Self; Merck Sharp &amp; Dohme Corp., National Institute of Diabetes and Digestive and Kidney Diseases. Research Support; Spouse/Partner; National Institute of Diabetes and Digestive and Kidney Diseases. A. Nasiri: Employee; Spouse/Partner; Medtronic. G. Cline: None. X. Zhang: None. J. Lee: None. B.T. Hubbard: None.

251 Oxidation of energy substrates in tissues of Largemouth bass (Micropterus salmoides)

Abstract Because of growing interest in producing Largemouth bass (HMB) as a source of high-quality protein for human consumption worldwide, it is imperative to understand the metabolism of nutrients (including amino acids and carbohydrate) in this aquatic animal. The present study tested the hypothesis that amino acids are oxidized at a higher rate than carbohydrates (e.g., glucose) and fatty acids (e.g., palmitate) to provide ATP for tissues of LMB fed a 45%-crude protein diet. The liver, intestine, kidney, and skeletal muscle were isolated from juvenile LMB and incubated at 26 °C (the body temperature of LMB) for 2 h in 1 ml of oxygenated Krebs–Henseleit bicarbonate buffer (pH 7.4) containing a mixture of nutrients (2 mM glutamate, 2 mM glutamine, 2 mM aspartate, 2 mM alanine, 2 mM leucine, 5 mM glucose, and 2 mM palmitate). The rate of oxidation of each energy substrate was determined by using [U-14C]-labeled glutamate, glutamine, aspartate, alanine, leucine, glucose, or palmitate and collecting 14CO2 from each tracer. Results indicated that aspartate, glutamate and glutamine were extensively oxidized in all the four tissues and contributed to 67% of total ATP production. Glutamate contributed to more ATP than glutamine in the intestine, whereas similar amounts of ATP were produced from glutamate and glutamine in the liver, kidneys and skeletal muscle. In all the four tissues, rates of oxidation of alanine, leucine, palmitate and glucose were low and each of those nutrients contributed to &amp;lt; 10% of total ATP production. Together, the oxidation of aspartate, glutamate, glutamine, alanine plus leucine provided 82–85% of total ATP for the liver, intestine, kidney, and skeletal muscle. We conclude that amino acids, rather than glucose and long-chain fatty acids, are the primary energy substrates in the major tissues of Largemouth bass.

253 Glutamate and glutamine are the major metabolic fuels in enterocytes of suckling piglets

Abstract Glutamate and glutamine are known to be important energy substrates in pig enterocytes, and aspartate has also been reported to be extensively catabolized by the rat small intestine. However, little is known about the relative role of amino acids, glucose and fatty acids in ATP production by enterocytes. In the present study, enterocytes isolated from 0-, 7-, 14- and 21-day-old piglets were used to determine the rates of oxidation of amino acids, fatty acids and glucose. Enterocytes were incubated at 37oC for 30 min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and one of the following: 0.5–5 mM L-[U-14C]glutamate, 0.5–5 mM L-[U-14C]glutamine, 0.5–5 mM L-[U-14C]aspartate, 0.5–5 mM L-[U-14C]alanine, 0.5–2 mM L-[U-14C]palmitate, D-[U-14C]glucose, 0.5–5 mM [U-14C]propionate, or 0.5–5 mM [1-14C]butyrate. At the end of the incubation, 14CO2 produced from each 14C-labeled substrate was collected. Rates of oxidation of each substrate in enterocytes from all age groups of piglets increased (P &amp;lt; 0.01) progressively with increasing its extracellular concentrations. The rates of oxidation of glutamate, glutamine, aspartate, glucose by enterocytes from 0- to 21-day-old pigs were much greater (P &amp;lt; 0.01) than those for the same concentrations of alanine, palmitate, propionate, and butyrate. In cells from all age groups of piglets, rates of oxidation of, and ATP production from 5 mM glutamate or 5 mM glutamine were greater (P &amp;lt; 0.01) than those from 5 mM glucose and aspartate. Oxidation of alanine, propionate, butyrate and palmitate by enterocytes was limited. At each postnatal age, the oxidation of glutamate and glutamine produced more ATP than any other substrates. Our results indicated that glutamate and glutamine are the major metabolic fuels in enterocytes of 0- to 21-day-old pigs. Because of limited uptake of arterial glutamate by enterocytes, dietary glutamate is essential to the integrity and function of the pig small intestine.

Ex Vivo Method to Simultaneously Evaluate Glucose Utilization, Uptake, and Production in Rat Liver.

A novel ex vivo method to simultaneously evaluate hepatic glucose utilization, uptake, and production was developed in rats. The right lateral lobe of the liver was perfused with Krebs-Henseleit bicarbonate buffer containing 5 mmol/L uniformly labeled 13C-glucose ([U-13C]-glucose). The whole glucose concentration in the perfusate was measured by colorimetric assay, and the concentrations of [U-12C]-glucose (natural isotope) or [U-13C]-glucose were estimated on the basis of the abundance ratio of [U-12C]-glucose or [U-13C]-glucose, which were measured by GC-MS. The difference in whole glucose and [U-13C]-glucose concentrations between the baseline and effluent perfusate represents hepatic glucose utilization and glucose uptake, respectively. The [U-12C]-glucose concentration in the effluent perfusate corresponds to hepatic glucose production. With this method, we clarified the precise mechanism that underlies the hepatic impairment of diabetic animals and pharmacological effects of anti-diabetic agents. Thus, this method is useful for the pathophysiological and pharmacological research of type 2 diabetes.

Teucrium polium L. Improved Heart Function and Inhibited Myocardial Apoptosis in Isolated Rat Heart Following Ischemia-Reperfusion Injury.

ObjectivesMyocardial reperfusion is the only logical cure for ischemic heart disease. However, ischemic-reperfusion (I/R) injury is one of the underlying factors facilitating and accelerating the apoptosis in the myocardium. This study set to investigate the impact of Teucrium polium (TP) hydro-alcoholic extract on I/R induced apoptosis in the isolated rat heart.MethodsIsolated rat hearts were classified into six groups. The control samples were subjected to 80 min of perfusion with Krebs–Henseleit bicarbonate (KHB) buffer; in control-ischemia group, after primary perfusion (20 min) the hearts were exposed to global ischemia (20 min) and reperfusion (40 min). Pretreated groups were perfused with 500 μM of vitamin C and various TP concentrations (0.5, 1, 2 mg/ml) for 20 min, and then the hearts were exposed to ischemia and reperfusion for 20 min and 40 min, respectively. Cardiodynamic parameters including rate pressure product (RPP), heart rate (HR), the maximum up/down rate of left ventricular pressure (±dp/dt), left ventricular developed pressure (LVDP), and coronary artery flow (CF) were achieved from Lab Chart software data. The Bax and BCl-2 gene expressions were measured in heart samples.ResultsHearts treated with TP extract and vit C represented a meaningful improvement in cardiac contractile function and CF. The overexpression of Bcl-2, downregulation of Bax, and improvement of apoptotic index (Bax/Bcl-2) were observed in pretreated TP extract and vit C hearts.ConclusionThe TP extract was found to ameliorate the cardiac function in the reperfused myocardium. Also, it can hinder apoptotic pathways causing cardioprotection.

Cytoprotective Effects of Mesenchymal Stem Cells During Liver Transplantation from Donors After Cardiac Death in Rats

BackgroundLiver transplantation from donors after cardiac death (DCD) might increase the pool of available organs. Recently, some investigators reported the potential use of mesenchymal stem cells (MSCs) to improve the outcome of liver transplantation from DCD. The aim of this study was to evaluate the cytoprotective effects and safety of MSC transplantation on liver grafts from DCD. MethodsRats were divided into 4 groups (n = 5) as follows: 1. the heart-beating group, in which liver grafts were retrieved from heart-beating donors; 2. the DCD group, in which liver grafts were retrieved from DCD that had experienced apnea-induced agonal conditions; 3. the MSC-1 group, and 4. the MSC-2 group, in which liver grafts were retrieved as with the DCD group, but were infused MSCs (2.0 × 105 or 1.0 × 106, respectively). The retrieved livers were perfused with oxygenated Krebs-Henseleit bicarbonate buffer (37°C) through the portal vein for 2 hours after 6 hours of cold preservation. Perfusate, bile, and liver tissues were then investigated. ResultsBile production in the MSC-2 group was significantly improved compared with that in the DCD group. Based on histologic findings, narrowing of the sinusoidal space in the both MSC groups was improved compared with that in the DCD group. ConclusionsMSCs could protect the function of liver grafts from warm ischemia-reperfusion injury and improve the viability of DCD liver grafts. In addition, we found that the infusion of 1.0 × 106 MSCs does not obstruct the hepatic sinusoids of grafts from DCD.

Donor Treatment With a Hypoxia-Inducible Factor-1 Agonist Prevents Donation After Cardiac Death Liver Graft Injury in a Rat Isolated Perfusion Model.

The protective role of hypoxia-inducible factor-1 (HIF-1) against liver ischemia-reperfusion injury has been well proved. However its role in liver donation and preservation from donation after cardiac death (DCD) is still unknown. The objective of this study was to test the hypothesis that pharmaceutical stabilization of HIF-1 in DCD donors would result in a better graft liver condition. Male SD rats (6 animals per group) were randomly given the synthetic prolyl hydroxylase domain inhibitor FG-4592 (Selleck, 6 mg/kg of body weight) or its vehicle (dimethylsulfoxide). Six hours later, cardiac arrest was induced by bilateral pneumothorax. Rat livers were retrieved 30 min after cardiac arrest, and subsequently cold stored in University of Wisconsin solution for 24 h. They were reperfused for 60 min with Krebs-Henseleit bicarbonate buffer in an isolated perfused liver model, after which the perfusate and liver tissues were investigated. Pretreatment with FG-4592 in DCD donors significantly improved graft function with increased bile production and synthesis of adenosine triphosphate, decreased perfusate liver enzyme release, histology injury scores and oxidative stress-induced cell injury and apoptosis after reperfusion with the isolated perfused liver model. The beneficial effects of FG-4592 is attributed in part to the accumulation of HIF-1 and ultimately increased PDK1 production. Pretreatment with FG-4592 in DCD donors resulted in activation of the HIF-1 pathway and subsequently protected liver grafts from warm ischemia and cold-stored injury. These data suggest that the pharmacological HIF-1 induction may provide a clinically applicable therapeutic intervention to prevent injury to DCD allografts.

Amino acids are major energy substrates for tissues of hybrid striped bass and zebrafish.

Fish generally have much higher requirements for dietary protein than mammals, and this long-standing puzzle remains unsolved. The present study was conducted with zebrafish (omnivores) and hybrid striped bass (HSB, carnivores) to test the hypothesis that AAs are oxidized at a higher rate than carbohydrates (e.g., glucose) and fatty acids (e.g., palmitate) to provide ATP for their tissues. Liver, proximal intestine, kidney, and skeletal muscle isolated from zebrafish and HSB were incubated at 28.5°C (zebrafish) or 26°C (HSB) for 2h in oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5mM D-glucose) containing 2mM L-[U-14C]glutamine, L-[U-14C]glutamate, L-[U-14C]leucine, or L-[U-14C]palmitate, or a trace amount of D-[U-14C]glucose. In parallel experiments, tissues were incubated with a tracer and a mixture of unlabeled substrates [glutamine, glutamate, leucine, and palmitate (2mM each) plus 5mM D-glucose]. 14CO2 was collected to calculate the rates of substrate oxidation. In the presence of glucose or a mixture of substrates, the rates of oxidation of glutamate and ATP production from this AA by the proximal intestine, liver, and kidney of HSB were much higher than those for glucose and palmitate. This was also true for glutamate in the skeletal muscle and glutamine in the liver of both species, glutamine in the HSB kidney, and leucine in the zebrafish muscle, in the presence of a mixture of substrates. We conclude that glutamate plus glutamine plus leucine contribute to ~80% of ATP production in the liver, proximal intestine, kidney, and skeletal muscle of zebrafish and HSB. Our findings provide the first direct evidence that the major tissues of fish use AAs (mainly glutamate and glutamine) as primary energy sources instead of carbohydrates or lipids.

Resveratrol Prevents Warm Ischemia–Reperfusion Injury in Liver Grafts From Non–Heart-Beating Donor Rats

BackgroundSuccessful liver transplantation from non–heart-beating donors (NHBDs) might enlarge donor source. Some studies have reported that resveratrol (RES), an activator of sirtuins, has cytoprotective effects on ischemia-reperfusion (I/R) injury. The purpose of this study was to investigate the effects of RES on warm I/R injury in rats. MethodsMale Wister rats were divided into 5 groups: (1) the heart-beating (HB) group, whose livers were retrieved from HB donors; (2) the NHB group, whose livers were retrieved under apnea-induced NHB conditions; (3) the ethanol group, retrieved in the same manner as the NHB group with ethanol (10 μL) as a solvent; (4) the RES-1 group, retrieved in the same manner as the NHB group and pretreated with RES (0.4 mg/kg, dissolved in 10 μL ethanol); and (5) the RES-2 group, retrieved in the same manner as the NHB group and pretreated with RES (2 mg/kg, dissolved in 10 μL ethanol). The resected livers were perfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after 6 hours of cold preservation, after which the perfusate and liver tissues were investigated. ResultsThe bile production, portal vein flow volume, tumor necrosis factor-α level, and adenosine triphosphate level in the RES-2 group were significantly improved compared with in the NHB group. Histology revealed numerous well-preserved sinusoidal endothelial cells in the RES-2 group. ConclusionsRES might reduce warm I/R injury and improve the viability of liver grafts from NHBDs. We considered that this method may represent a promising approach for clinical liver transplantation from NHBDs.

Pharmacological study of the mechanisms involved in the vasodilator effect produced by the acute application of triiodothyronine to rat aortic rings.

A relationship between thyroid hormones and the cardiovascular system has been well established in the literature. The present in vitro study aimed to investigate the mechanisms involved in the vasodilator effect produced by the acute application of 10-8–10-4 M triiodothyronine (T3) to isolated rat aortic rings. Thoracic aortic rings from 80 adult male Wistar rats were isolated and mounted in tissue chambers filled with Krebs-Henseleit bicarbonate buffer in order to analyze the influence of endothelial tissue, inhibitors and blockers on the vascular effect produced by T3. T3 induced a vasorelaxant response in phenylephrine-precontracted rat aortic rings at higher concentrations (10-4.5–10-4.0 M). This outcome was unaffected by 3.1×10-7 M glibenclamide, 10-3 M 4-aminopyridine (4-AP), 10-5 M indomethacin, or 10-5 M cycloheximide. Contrarily, vasorelaxant responses to T3 were significantly (P<0.05) attenuated by endothelium removal or the application of 10-6 M atropine, 10-5 M L-NG-nitroarginine methyl ester (L-NAME), 10-7 M 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10-6 M (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i](1,6)benzodiazocine-10-carboxylic acid, methyl ester KT 5823, 10-2 M tetraethylammonium (TEA), or 10-7 M apamin plus 10-7 M charybdotoxin. The results suggest the involvement of endothelial mechanisms in the vasodilator effect produced by the acute in vitro application of T3 to rat aortic rings. Possible mechanisms include the stimulation of muscarinic receptors, activation of the NO-cGMP-PKG pathway, and opening of Ca2+-activated K+ channels.

Effects of Syzygium aromaticum‐derived oleanolic acid on glucose transport and glycogen synthesis in the rat small intestine (丁香衍生物齐墩果酸对大鼠小肠中的葡萄糖转运以及糖原合成的影响)

AbstractBackground: In the present study, we investigated the effects of oleanolic acid (OA), which has hypoglycemic properties, on glucose transport and glycogen synthesis in the small intestine, an organ that secretes enzymes involved in carbohydrate metabolism.Methods: The OA was isolated from Syzygium aromaticum ethyl acetate‐soluble fractions followed by recrystallization with ethanol. It was diluted to required concentrations in freshly prepared dimethyl sulfoxide (2 mL) and normal saline (19 mL) before being administered to rats (p.o.). Glycogen concentrations were determined in isolated small intestines from fasted and non‐fasted non‐diabetic and streptozotocin‐diabetic rats after 18 h treatment with 80 mg/kg, p.o., OA or standard hypoglycemic drugs (i.e. 100 μg/kg, s.c., insulin; 500 mg/kg, p.o., metformin). In a separate series of experiments, the effects of 30‐min incubation with graded concentrations of OA (0.82–6.56 mmol/L) on d‐glucose were evaluated by monitoring changes in glucose concentrations inside and outside of intestinal sacs isolated from fasted, non‐diabetic rats and mounted in an organ bath containing Krebs–Henseleit bicarbonate buffer.Results: All in vivo treatments increased the glycogen concentration in rat small intestine, although the effects of metformin treatment in non‐fasted diabetic rats failed to reach statistical significance. In vitro, both OA (1.64–6.56 mmol/L) and phlorizin (10−5–10−3 mol/L) decreased glucose transport from the mucosa to the serosa.Conclusion: The data suggest that OA may be a potential alternative drug treatment for postprandial hyperglycemia because of its inhibition of glucose uptake across the small intestine and its concomitant conversion of glucose to glycogen in the intestinal wall.

Journal-related Activities and Other Special Activities at the 2012 American Society of Anesthesiologists Annual Meeting

Journal-related Activities and Other Special Activities at the 2012 American Society of Anesthesiologists Annual Meeting

Blood Modulates the Kinetics of Reactive Oxygen Release in Pancreatic Ischemia-Reperfusion Injury

Reason for the unsuccessful use of antioxidants in transplantation might be the unknown kinetics of reactive oxygen species (ROS) release. In this study, we compared the kinetics of ROS release from rat pancreata in the presence and absence of blood. In vivo, ischemia-reperfusion injury (IRI) was induced in pancreata of male Wistar rats by occlusion of the arterial blood supply for 1 or 2 hours. In vitro, isolated pancreata were single-pass perfused with Krebs-Henseleit bicarbonate solution. Reactive oxygen species were quantified by electron spin resonance spectroscopy using CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) as spin label. Thiols (glutathione), nicotinamide adenine dinucleotide phosphate-oxidase activity, myeloperoxidase activity, and adenosine triphosphate content were measured. During reperfusion, an increase in IRI-induced ROS in arterial blood was noted after 2 hours of warm ischemia. In sharp contrast, ROS release was immediate and short lived in blood-free perfused organs. The degree of tissue damage correlated with nicotinamide adenine dinucleotide phosphate-oxidase activity and adenosine triphosphate content. Antioxidative capacity of tissues was reduced. Electron spin resonance spectroscopy in conjunction with spin labels allows for the detection of ROS kinetics in pancreatic IRI. Reactive oxygen species kinetics are dependent on the length of the ischemic period and the presence or absence of blood.