Machine perfusion at 20°C reduces preservation damage to livers from non-heart beating donors

We previously reported that machine perfusion (MP) performed at 20 °C enhanced the preservation of steatotic rat livers. Here, we tested whether rat livers retrieved 30 min after cardiac arrest (NHBDs) were better protected by MP at 20 °C than with cold storage. We compared the recovery of livers from NHBDs with organs obtained from heart beating donors (HBDs) preserved by cold storage. MP technique: livers were perfused for 6 h with UW-G modified at 20 °C. Cold storage: livers were perfused in situ and preserved with UW solution at 4 °C for 6 h. Both MP and cold storage preserved livers were reperfused with Krebs-Heinselet buffer (2 h at 37 °C). AST and LDH release and mitochondrial glutamate dehydrogenase (GDH) levels were evaluated. Parameters assessed included: bile production and biliary enzymes; tissue ATP; reduced and oxidized glutathione (GSH/GSSG); protein–SH group concentration. Livers preserved by MP at 20 °C showed significantly lower hepatic damage at the end of reperfusion compared with cold storage. GDH release was significantly reduced and bile production, ATP levels, GSH/GSSG and protein–SH groups were higher in livers preserved by MP at 20 °C than with cold storage. The best preserved morphology and high glycogen content was obtained with livers submitted to MP at 20 °C. Liver recovery using MP at 20 °C was comparable to recovery with HBDs. MP at 20 °C improves cell survival and gives a better-quality of preservation for livers obtained from NHBDs and may provide a new method for the successful utilization of marginal livers.     

Dipeptidylpeptidase-IV Activity and Expression Reveal Decreased Damage to the Intrahepatic Biliary Tree in Fatty Livers Submitted to Subnormothermic Machine-Perfusion Respect to Conventional Cold Storage

Graft steatosis is a risk factor for poor initial function after liver transplantation. Biliary complications are frequent even after normal liver transplantation. A subnormothermic machine perfusion (MP20) preservation procedure was developed by our group with high potential for reducing injury to hepatocytes and sinusoidal cells of lean and fatty livers respect to conventional cold storage (CS). We report the response of the biliary tree to CS or MP20, in lean and obese Zucker rat liver. Dipeptidylpeptidase-IV (DPP-IV), crucial for the inactivation of incretins and neuropeptides, was used as a marker. Liver morphology and canalicular network of lean livers were similar after CS/reperfusion or MP20/reperfusion. CS preservation of fatty livers induced serious damage to the parenchyma and to the canalicular activity/ expression of DPP-IV, whereas with MP20 the morphology and canalicular network were similar to those of untreated lean liver. CS and MP20 had similar effects on DPP-IV activity and expression in the upper segments of the intrahepatic biliary tree of fatty livers. DPP-IV expression was significantly increased after MP20 respect to CS or to the controls, both for lean and obese animals. Our data support the superiority of MP20 over CS for preserving fatty livers. Dipeptidylpeptidase-IV activity and expression reveal decreased damage to the intrahepatic biliary tree in fatty livers submitted to subnormothermic machine-perfusion respect to conventional cold storage.Key words: Dipeptidylpeptidase-IV, fatty liver, bile tract, cold storage, subnormothermic machine perfusion     

Altered alkaline phosphatase activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all putative roles ascribed to the enzyme

Biliary complications often lead to acute and chronic liver injury after orthotopic liver transplantation (OLT). Bile composition and secretion depend on the integrated action of all the components of the biliary tree, starting from hepatocytes. Fatty livers are often discarded as grafts for OLT, since they are extremely vulnerable to conventional cold storage (CS). However, the insufficiency of donors has stimulated research to improve the usage of such marginal organs as well as grafts. Our group has recently developed a machine perfusion system at subnormothermic temperature (20°C; MP20) that allows a marked improvement in preservation of fatty and even of normal rat livers as compared with CS. We sought to evaluate the response of the biliary tree of fatty liver to MP20, and a suitable marker was essential to this purpose. Alkaline phosphatase (AlkP, EC 3.1.3.1), frequently used as marker of membrane transport in hepatocytes and bile ducts, was our first choice. Since no histochemical data were available on AlkP distribution and activity in fatty liver, we have first settled to investigate AlkP activity in the steatotic liver of fatty Zucker rats (fa/fa), using as controls lean Zucker (fa/+) and normal Wistar rats. The AlkP reaction in Wistar rats was in accordance with the existing data and, in particular, was present in bile canaliculi of hepatocytes in the periportal region and midzone, in the canals of Hering and in small bile ducts but not in large bile ducts. In lean ZR liver the AlkP reaction in Hering canals and small bile ducts was similar to Wistar rat liver but hepatocytes had lower canalicular activity and besides presented moderate basolateral reaction. The difference between lean Zucker and Wistar rats, both phenotypically normal animals, could be related to the fact that lean Zucker rats are genotypically heterozygous for a recessive mutated allele. In fatty liver, the activity in ductules and small bile ducts was unchanged, but most hepatocytes were devoid of AlkP activity with the exception of clusters of macrosteatotic hepatocytes in the mid-zone, where the reaction was intense in basolateral domains and in distorted canaliculi, a typical pattern of cholestasis. The interpretation of these data was hindered by the fact that the physiological role of AlkP is still under debate. In the present study, the various functions proposed for the role of the enzyme in bile canaliculi and in cholangiocytes are reviewed. Independently of the AlkP role, our data suggest that AlkP does not seem to be a reliable marker to study the initial step of bile production during OLT of fatty livers, but may still be used to investigate the behaviour of bile ductules and small bile ducts.     

Decreased apoptosis in fatty livers submitted to subnormothermic machine-perfusion respect to cold storage

Machine perfusion at subnormothermic temperature (20°C), MP20, was developed by Vairetti et al. and showed to afford a better preservation of fatty livers respect to traditional cold storage (CS) in terms of enzyme release into the perfusate and bile, glycogen stores, energy charge and oxidative stress. Here we investigated whether it also caused decreased cell death by apoptosis. Fatty and lean Zucker rats were submitted to MP20 or CS for 6 h and reperfused normothermically for 2 h. Apoptotic cells were revealed by immunohistochemistry of activated caspase-3 and M30 (new epitope on CK18 degraded by caspase-3) and by the TUNEL assay. Portal pressure was also determined. A statistically significant reduction of hepatocyte apoptosis, but especially of sinusoidal cells was determined for fatty livers submitted to MP20 respect to CS. Portal pressure was significantly lower after MP20 respect to CS. The reduction of sinusoidal cell death by apoptosis without need for anti-apoptotic therapies appears particularly positive since apoptotic sinusoidal cells hinder microcirculation in the sinusoids and are thrombogenic. These results further confirm the potential of MP20 for preserving fatty livers that would be otherwise discarded as grafts, and thus for increasing the donor pool for liver transplantation.     

The influence of storage temperature during machine perfusion on preservation quality of marginal donor livers

Background

Although non-heart-beating donors have the potential to increase the number of available organs, the livers are used very seldom because of the risk of primary non-function. There is evidence that machine perfusion is able to improve the preservation of marginal organs, and therefore we evaluated in our study the influence of the perfusate temperature during oxygenated machine perfusion on the graft quality.

Methods

Livers from male Wistar rats were harvested after 60-min warm ischemia induced by cardiac arrest. The portal vein was cannulated and the liver flushed with Lifor® (Lifeblood Medical, Inc.) organ preservation solution for oxygenated machine perfusion (MP) at 4, 12 or 21 °C. Other livers were flushed with HTK and stored at 4 °C by conventional cold storage (4 °C-CS). Furthermore two groups with either warm ischemic damage only or without any ischemic damage serve as control groups. After 6 h of either machine perfusion or cold storage all livers were normothermic reperfused with Krebs–Henseleit buffer, and functional as well as structural data were analyzed.

Results

Contrary to livers stored by static cold storage, machine perfused livers showed independently of the perfusate temperature a significantly decreased enzyme release of hepatic transaminases (ALT) during isolated reperfusion. Increasing the machine perfusion temperature to 21 °C resulted in a marked reduction of portal venous resistance and an increased bile production.

Conclusions

Oxygenated machine perfusion improves viability of livers after prolonged warm ischemic damage. Elevated perfusion temperature of 21 °C reconstitutes the hepatic functional capacity better than perfusion at 4 or 12 °C.     

Stimulatory Effect of Leptin on Nitric Oxide Production Is Impaired in Dietary‐Induced Obesity

Objective: We investigated the effect of leptin on nitric oxide production in lean and rats made obese by a high‐calorie diet. Research Methods and Procedures: The animals were placed in metabolic cages, and urine was collected in 2‐hour periods after leptin (1 mg/kg intraperintoneally) or vehicle administration. Blood was obtained 0.5, 1, 2, 4, or 6 hours after injection. Results: Leptin had no effect on systolic blood pressure in either lean or obese animals. Plasma concentration of NO metabolites (nitrites + nitrates, NO_x) increased in lean rats by 31.5%, 58.0%, and 27.9% at 1, 2, and 4 hours after leptin injection, respectively. In the obese group, plasma NO_x increased only at 2 hours (+36.5%). Leptin increased urinary NO_x excretion by 31.8% in the first 2‐hour period after injection in lean but not in obese rats. In lean animals, leptin elevated plasma cyclic 3′, 5′‐guanosine monophosphate (cGMP) at 1, 2, and 4 hours by 35.3%, 96.3%, and 57.3%, respectively. In the obese group, plasma cGMP was higher only at 2 and 4 hours (+44.6% and +32.1%, respectively). Urinary excretion of cGMP increased in lean animals by 67.1% in the first period and by 50.4% in the second period. In the obese group, leptin induced a 53.9% increase in urinary cGMP excretion only in the first 2‐hour period. Discussion: The stimulatory effect of leptin on NO production is impaired in dietary‐induced obesity; however, leptin does not increase blood pressure in obese animals, suggesting that other NO—independent depressor mechanisms are stimulated.     

Changes in laminin immunoreactivity as a marker for the state of liver preservation

Summary In order to examine the role of the extracellular matrix glycoprotein laminin as a marker for the preservation of liver tissue, dog livers were perfused and then preserved for 5 min, 1, 2, 4, 6, 8, 10, 12, 22 and 26 hours with HTK (histidine-tryptophan-ketoglutarate) solution at 5°C and at 25°C and with UW (University of Wisconsin) solution at 5°C. The tissue was processed for the immunohistochemical demonstration of laminin using an anti-P1 and an anti-E8 antibody. The peroxidase-antiperoxidase method was used for the visualization of the immunohistochemical reaction. At the beginning of the preservation, immunostaining was observed for both fragments of laminin around bile ducts and blood vessels of the portal spaces, under all preservation conditions. Clear immunostaining was also visible in the wall of the terminal arterioles located between the liver lobules. In the 5°C-preserved tissues, immunostaining for both laminin fragments occurred for preservation times between 4 and 6 hours in the form of isolated perisinusoidal deposits at the transition point where the sinusoids sprout from the terminal venules. In the 25°C-preserved tissue, such a staining pattern was already visible after 1 to 2 hours, preservation time. Our results show that the occurrence of laminin immunoreactivity in the sinusoids can be taken as a marker for the state of liver preservation. A hypothesis for the presence and the role of this glycoprotein in the perisinusoidal space is presented.     

Gaseous oxygen persufflation or oxygenated machine perfusion with Custodiol-N for long-term preservation of ischemic rat livers?

The aim of the present study was to evaluate the potential benefit of two different techniques for the provision of tissue aerobiosis upon cold preservation of marginal livers from non-heart beating donors using a recently developed improved preservation solution.Rat livers were harvested 30 min after cardiac arrest, flushed via the portal vein and cold-stored in HTK or modified HTK-solution (Custodiol-N) for 18 h at 4 °C. Other organs were flushed with Custodiol-N and subjected to aerobic conditions by either vascular systemic oxygen persufflation (VSOP) of the cold stored organ or hypothermic machine perfusion (HMP) with oxygenated Custodiol-N. Viability of the livers was assessed after 18 h of preservation by warm reperfusion in vitro for 120 min.Free radical mediated lipid peroxidation was significantly abrogated by the use of Custodiol-N in all groups compared with HTK. Custodiol-N improved enzyme leakage upon reperfusion and histological integrity, but had no impact on functional recovery (bile production, energetic status). However, VSOP further minimized enzyme release during the whole reperfusion period, led to a rise in hepatic bile production and enhanced recovery of energy charge (p < 0.05, resp. vs Custodiol-N). Histological appearance was concordantly improved in VSOP. During the first 45 min of reperfusion, leakage of ALT and LDH was also reduced by MP but deteriorated thereafter and became significantly higher compared to Custodiol-N at the end of the experiment. In conclusion, the results of the present study recommend the use of gaseous oxygen persufflation to improve tissue integrity and functional recovery of predamaged livers.     

Effect of drug treatment on liver-slice function following 72-hour hypothermic perfusion

The viability of hypothermically perfused dog liver was evaluated with a tissue-slice technique. After being preserved for 72 hr, slices of liver were incubated at 30 degrees C for as long as 2 hr; then water content, K+/Na+ ratio, and ATP concentration were measured. Dog livers were assigned to the following experimental groups: Group 1 (no preservation; control); Group 2 (livers preserved for 72 hr); Group 3 (donor animals pretreated with 3.5 mg/kg of chlorpromazine (CPZ) and 20 mg/kg of methylprednisolone (MP), and livers preserved for 72 hr); Group 4 (livers pretreated with 2-deoxycoformycin (2-DOC), 50 mg/liter, and preserved for 72 hr); and Group 5 (combination of Group 3 and Group 4 treatments). Livers in Groups 2, 3, and 4 lost K+ during preservation, and the mean K+/Na+ ratio significantly decreased from a control value of 4.2 +/- 0.4 to 1.5-1.9 (P less than 0.05). Group 5 livers did not lose K+; mean K+/Na+ ratio was 3.9 +/- 0.5. Fresh livers (no preservation) rapidly reaccumulated K+ when the tissue slices were incubated for 2 hr at 30 degrees C; mean K+/Na+ ratio was 3.7 +/- 0.5. Tissue slices from Group 2 livers (72 hr preservation), and livers pretreated with CPZ-MP (Group 3) or pretreated with 2-DOC (Group 4) did not significantly reaccumulate K+ at 30 degrees C; mean K+/Na+ ratio was 1.7-2.1. Only slices prepared from liver pretreated with both CPZ-MP and 2-DOC reaccumulated K+; mean K+/Na+ ratio was 4.6 +/- 1.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of exercise and obesity on skeletal muscle amino acid uptake

The genetically obese Zucker rat has a reduced capacity to deposit dietary protein in skeletal muscle. To determine whether amino acid uptake by muscle of obese Zucker rats is impaired, soleus strip (SOL) and epitrochlearis (EPI) muscles from 10-wk-old lean and obese Zucker rats were studied in vitro by use of [14C]alpha-aminoisobutyric acid (AIB). Muscles from fasted rats were incubated under basal conditions at rest or after a 1-h treadmill run at 8% grade. To equate total work completed, lean and obese rats ran at 27 and 20 m/min, respectively. Muscles were pinned at resting length, preincubated for 30 min at 37 degrees C in Krebs-Ringer bicarbonate buffer containing 5 mM glucose under 95% O2-5% CO2, and then incubated up to 3 h in Krebs-Ringer bicarbonate with 0.5 mM AIB, [14C]AIB, and [3H]inulin as a marker of extracellular fluid. Basal AIB uptake in EPI and SOL from obese rats was significantly reduced by 40 and 30% (P less than 0.01), respectively, compared with lean rats. For both lean and obese rats, exercise increased (P less than 0.05) basal AIB uptake in EPI and SOL, but the relative increases were greater in the obese rats (EPI 54% and SOL 71% vs. EPI 32% and SOL 37%). These results demonstrate that genetically obese Zucker rats have reduced basal skeletal muscle amino acid uptake and suggest that physical inactivity may partially contribute to this defect.     

Effects of method of preservation on functions of livers from fed and fasted rabbits

Livers from fed, fasted (48 h) and glucose-fed rabbits were preserved for 24 and 48 h by either simple cold storage (CS) or continuous machine perfusion (MP) with the University of Wisconsin preservation solutions. After preservation liver functions were measured by isolated perfusion of the liver (at 37 degrees C) for 2 h. Fasting caused an 85% reduction in the concentration of glycogen in the liver but no change in ATP or glutathione. Glucose feeding suppressed the loss of glycogen (39% loss). After 24 h preservation by CS livers from fed or fasted animals were similar including bile production (6.2 +/- 0.5 and 5.6 +/- 0.4 ml/2 h, 100 g, respectively), hepatocellular injury (LDH release = 965 +/- 100 and 1049 +/- 284 U/liter), and concentrations of ATP (1.17 +/- 0.15 and 1.18 +/- 0.04 mumol/g, glutathione (1.94 +/- 0.51 and 2.35 +/- 0.26 mumol/g, respectively), and K:Na ratio (6.7 +/- 1.0 and 7.7 +/- 0.5, respectively). After 48 h CS livers from fed animals were superior to livers from fasted animals including significantly more bile production (5.0 +/- 0.9 vs 2.0 +/- 0.3 ml/2 h, 100 g), less LDH release (1123 +/- 98 vs 3701 +/- 562 U/liter), higher concentration of ATP (0.50 +/- 0.16 vs 0.33 +/- 0.07 mumol/g) and glutathione (0.93 +/- 0.14 vs 0.30 +/- 0.13 mumol/g), and a larger K:Na ratio (7.4 vs 1.5). Livers from fed animals were also better preserved than livers from fasted animals when the method was machine perfusion. The decrease in liver functions in livers from fasted animals preserved for 48 h by CS or MP was prevented by feeding glucose. Glucose feeding increased bile formation after 48 h CS preservation from 2.0 +/- 0.3 (fasted) to 6.9 +/- 1.2 ml/2 h, 100 g; LDH release was reduced from 3701 +/- 562 (fasted) to 1450 +/- 154 U/liter; ATP was increased from 0.33 +/- 0.07 (fasted) to 1.63 +/- 0.18 mumol/g; glutathione was increased from 0.30 +/- 0.01 (fasted) to 2.17 +/- 0.30 mumol g; and K:Na ratio was increased from 1.5 +/- 0.9 to 5.3 +/- 1.0. This study shows that the nutritional status of the donor can affect the quality of liver preservation. The improvement in preservation by feeding rabbits only glucose suggests that glycogen is an important metabolite for successful liver preservation. Glycogen may be a source for ATP synthesis during the early period of reperfusion of preserved livers.     

Impaired nitric oxide synthase-dependent dilatation of cerebral arterioles in type II diabetic rats

The goals of this study were to determine the effects of type II diabetes mellitus on nitric oxide synthase-dependent responses of cerebral arterioles and on endothelial nitric oxide synthase (eNOS) protein in cerebral arterioles. We examined dilatation of cerebral (pial) arterioles in 13-15 week old male lean and diabetic obese Zucker rats in response to nitric oxide synthase-dependent agonists (acetylcholine and adenosine diphosphate (ADP)) and a nitric oxide synthase-independent agonist (nitroglycerin). We found that acetylcholine (10 microM) increased cerebral arteriolar diameter by 10 +/- 3% (mean +/- SE) in lean Zucker rats, but by only 2 +/- 2% in diabetic obese Zucker rats (p<0.05). In addition, ADP (100 microM) increased cerebral arteriolar diameter by 20 +/- 2% in lean Zucker rats, but by only 8 +/- 2% in diabetic obese Zucker rats (p<0.05). In contrast, nitroglycerin produced similar vasodilatation in lean and diabetic obese Zucker rats. Thus, impaired dilatation of cerebral arterioles in diabetic obese Zucker rats is not related to non-specific impairment of vasodilatation. Following these functional studies, we harvested cerebral microvessels for Western blot analysis of eNOS protein. We found that eNOS protein was significantly higher in diabetic obese Zucker rats than in lean Zucker rats (p<0.05). Thus, type II diabetes mellitus impairs nitric oxide synthase-dependent responses of cerebral arterioles. In addition, eNOS protein from cerebral blood vessels is increased in diabetic obese Zucker rats.     

Responses and song pattern copying of Omega-type I-neurons in the cricket,Gryllus bimaculatus,at different prothoracic temperatures

Summary Omega-type I-neurons (ON/1) (Fig. 1A) were recorded intracellularly with the prothoracic ganglion kept at temperatures of either 8–9°, or 20–22° or 30–33 °C and the forelegs with the tympanal organs kept at ambient temperature (20–22 °C). The neurons were stimulated with synthetic calling songs (5 kHz carrier frequency) with syllable periods (SP in ms) varying between 20 and 100, presented at sound intensities between 40 and 80 dB SPL. The amplitude and duration of spikes as well as response latency decreased at higher temperatures (Figs. 1 B, 2, 6). At lower prothoracic temperatures (8–9 °C) the neuron's responses to songs with short SP (20 ms) failed to copy single syllables, or with moderate SP (40 ms) copied the syllable with low signal to noise ratio (Fig. 3). The auditory threshold of the ON/1 type neuron, when tested with the song model, was temperature-dependent. At 9° and 20 °C it was between 40 and 50 dB SPL and at 33 °C it was less than 40 dB SPL (Fig. 4). For each SP, the slope of the intensity-response function was positively correlated with temperature, however, at low prothoracic temperatures the slope was lower for songs with shorter SPs (Fig. 5). The poor copying of the syllabic structure of the songs with short SPs at low prothoracic temperatures finds a behavioral correlate because females when tested for phonotaxis on a walking compensator responded best to songs with longer SPs at a similar temperature.Abbreviations epsps excitatory postsynaptic potentials - ON/1 omega-type I-neuron - SP syllable period - SPL sound pressure level     

Dopamine improves hypothermic machine preservation of the liver

Background

Hypothermic machine preservation (HMP) is currently reconsidered as alternative to standard cold storage of organs from non-heart-beating donors. The present study was aimed at investigating the possible synergistic effect of HMP and the addition of dopamine to the circulating perfusate during preservation.

Methods

Cardiac arrest was induced in male Wistar rats (250–300 g) by phrenotomy. Thirty minutes later livers were flushed via the portal vein and subjected to 20 h of HMP at 5 ml/min at 4 °C. During HMP the preservation solution was equilibrated with 100% oxygen and dopamine was added at 0, 10, 50 or 100 μM (D0, D10, D50, D100; n = 6 resp.). Graft viability was assessed thereafter upon warm reperfusion in vitro for 2 h.

Results

During HMP, D50 and D100 significantly reduced hepatic release of ALT to about 50%. No influence of dopamine was found on vascular resistance, oxygen uptake or lactate production at any concentration. D50 significantly reduced enzyme release during reperfusion (∼50%), enhanced bile flow and oxygen consumption. D10 was less effective while D100 even rose enzyme release compared with D0.Enhanced oxygen free radical mediated lipid peroxidation (LPO), found in the tissue of D0 livers was significantly reduced by D50; D50 significantly abrogated molecular upregulation of vWillebrand factor upon reperfusion suggesting vascular protection of the endothelial cell.

Conclusion

Efficiency of HMP might be increased by stimulating livers with dopamine during ex vivo preservation, limiting vascular side effects and improving functional recovery upon early reperfusion.     

Resistance of hepatic glycogen to depletion in obese Zucker rats

Glycogen stores (liver and carcass) have been studied in lean and obese Zucker rats. The animals were submitted to one of three feeding conditions: ad libitum, a 48-h fast, or a 48-h fast and food ad libitum for 24 h, and to two environmental conditions, either thermoneutrality or an acute cold exposure (2 days at 4-7 degrees C). After a 2-day fast at 25 degrees C, the liver glycogen store was reduced by 45 times in the lean rats, while it was decreased by only 3 times in the obese rats. Under these conditions, the liver glycogen store was 45 times higher in the obese than in the lean rats. After 2 days in the cold, liver glycogen store was 4.4 times higher in obese rats than in lean rats. After a 2-day fast in the cold, the liver glycogen store in the obese rats was 30 times higher than in the lean rats. In comparison to fasting at thermoneutrality, fasting in the cold did not lead to a further reduction in hepatic glycogen in obese Zucker rats. The differences observed in the mobilization of the hepatic glycogen store between obese and lean rats have not been found in the mobilization of the carcass glycogen store. Drastic conditions, such as a 2-day fast in the cold, did not exhaust the glycogen store in obese Zucker rats. The present observations point out that obese Zucker rats cannot mobilize the entire hepatic glycogen store, as seen in lean control rats. The role of this abnormality in the high hyperlipogenesis that maintains the obese state is still to be evaluated.     

Altered Kidney CYP2C and Cyclooxygenase‐2 Levels Are Associated with Obesity‐Related Albuminuria

Objective: To determine cytochrome P450 (CYP450) and cyclooxygenase (COX) expression and metabolite regulation and renal damage in the early stages of obesity‐related hypertension and diabetes. Research Methods and Procedures: Obese and lean Zucker rats at 10 to 12 weeks of age were studied. Blood pressure was measured in the conscious state using radiotelemetry. Blood glucose levels and body weight were measured periodically. Protein expression of CYP450 and COX enzymes in the kidney cortex, renal microvessels, and glomeruli was studied. The levels of CYP450 and COX metabolites in urine were measured, and urinary albumin excretion, an indicator of kidney damage, was measured. Results: Body weight and blood glucose averaged 432 ± 20 grams and 105 ± 5 mg/dl, respectively, in obese Zucker rats as compared with 320 ± 8 grams and 91 ± 5 mg/dl, respectively, in age‐matched 10‐ to 12‐week‐old lean Zucker rats. Renal microvascular CYP4A and COX‐2 protein levels were increased 2.3‐ and 17.0‐fold, respectively, in obese Zucker rats. The protein expression of CYP2C11 and CYP2C23 was decreased 2.0‐fold in renal microvessels isolated from obese Zucker rats when compared with lean Zucker rats. The urinary excretion rate of thromboxane B₂ was increased significantly in obese Zucker as compared with lean Zucker rats (22.0 ± 1.8 vs. 13.4 ± 1.0 ng/d). Urinary albumin excretion, an index of kidney damage, was increased in the obese Zucker rat at this early age. Discussion: These results suggest that increased CYP4A and COX‐2 protein levels and decreased CYP2C11 and CYP2C23 protein levels occur in association with microalbuminuria during the onset of obesity‐related hypertension and type 2 diabetes.     

Nitric oxide concentrations in gas emanating from the tails of obese rats

Abstract

This study was undertaken to investigate the effects of oral L-arginine administration and exercising training on the NO concentration emanating from rat tail and NO_x in plasma. Obese (fa/fa) Zucker rats (n = 22) were divided into four groups: (1) oral L-arginine administration (A) (n = 6), (2) exercise training (E), (3) exercise training + L-arginine administration (E + A) (n = 5), and (4) non-exercise training + non-L-arginine administration (N) (n = 6). The control (+/+) Zucker rats (n = 22) were also divided into the same four groups. The body weight of the E + A and the A groups was significantly lower than that of the N group. The NO concentration emitted from the tail was higher in the L-arginine (E + A and A) groups than in the non-L-arginine (E and N) groups in both obese and control rats. Exercise training did not affect the skin gas NO concentration in either obese or control rats. Plasma NO_x concentrations in four obese rats were significantly higher than those observed in control rats. Exercise training did not influence the level of plasma NO_x in obese or control rats. In conclusion, this study confirmed that L-arginine administration increases the skin gas NO concentration and obesity increases the plasma NO_x level. The plasma NO_x concentrations were not affected by L-arginine administration or exercise training in obese or control rats.     

Cardiovascular and metabolic responses to fasting and thermoneutrality are conserved in obese Zucker rats

The primary purpose of the study was to test the hypothesis that reduced leptin signaling is necessary to elicit the cardiovascular and metabolic responses to fasting. Lean (Fa/?; normal leptin receptor; n = 7) and obese (fa/fa; mutated leptin receptor; n = 8) Zucker rats were instrumented with telemetry transmitters and housed in metabolic chambers at 23 degrees C (12:12-h light-dark cycle) for continuous (24 h) measurement of metabolic and cardiovascular variables. Before fasting, mean arterial pressure (MAP) was higher (MAP: obese = 103 +/- 3; lean = 94 +/- 1 mmHg), whereas oxygen consumption (VO(2): obese = 16.5 +/- 0.3; lean = 18.6 +/- 0.2 ml. min(-1). kg(-0.75)) was lower in obese Zucker rats compared with their lean controls. Two days of fasting had no effect on MAP in either lean or obese Zucker rats, whereas VO(2) (obese = -3.1 +/- 0.3; lean = -2.9 +/- 0.1 ml. min(-1). kg(-0.75)) and heart rate (HR: obese = -56 +/- 4; lean = -42 +/- 4 beats/min) were decreased markedly in both groups. Fasting increased HR variability both in lean (+1.8 +/- 0.4 ms) and obese (+2.6 +/- 0.3 ms) Zucker rats. After a 6-day period of ad libitum refeeding, when all parameters had returned to near baseline levels, the cardiovascular and metabolic responses to 2 days of thermoneutrality (ambient temperature 29 degrees C) were determined. Thermoneutrality reduced VO(2) (obese = -2.4 +/- 0.2; lean = -3.3 +/- 0.2 ml. min(-1). kg(-0.75)), HR (obese = -46 +/- 5; lean = -55 +/- 4 beats/min), and MAP (obese = -13 +/- 6; lean = -10 +/- 1 mmHg) similarly in lean and obese Zucker rats. The results indicate that the cardiovascular and metabolic responses to fasting and thermoneutrality are conserved in Zucker rats and suggest that intact leptin signaling may not be requisite for the metabolic and cardiovascular responses to reduced energy intake.     

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

Introduction

Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions.Methods: Human umbilical vein endothelial cells (HUVEC) were preserved at 4–37 °C for up to 20 h using Ringer's lactate, histidine–tryptophan–ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters.Results: HUVEC viability and barrier integrity was compromised at 4 °C regardless of the preservation solution. At temperatures above 20 °C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20 °C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20 °C except for ECGM.Conclusion: Optimal HUVEC preservation is achieved with UW up to 20 °C. Only ECGM maintains HUVEC viability at temperatures above 20 °C.     

Increased Expression of Complement Component C3 in the Plasma of Obese Zucker fa and LA/N fef Rats Compared with Their Lean Counterparts

Objectives : The objectives of this study were to determine whether there are differences in the electrophoretic profiles of plasma proteins from lean and obese rats and to identify a protein that was found to be more abundant in the plasma of obese rats. Research Methods and Procedures : Plasma proteins from lean and obese Zucker fa and LA/N/fa^frats were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of a band that was differentially expressed was determined by amino acid sequencing and Western blot analysis. Results : A band migrating approximately the same distance as the 116 kDa molecular weight marker was more prominent in plasma from obese rats than in plasma of lean rats. Partial sequencing of the peptide revealed that 17 of the first 18 amino acids at the amino terminus were identical with the corresponding residues in the α-chain of complement component C3. Western blot analysis confirmed the identity of the peptide as complement component C3. Complement C3 activity was measured using a hemolytic assay to determine whether there was a corresponding increase in the biological activity of this component in the serum of obese rats. Serum from obese rats was found to have 1.8 times as much complement component C3 activity as serum from lean rats. Discussion : Elevated levels of complement C3 in genetically obese rats may be relevant because increased amounts of C3 could serve as a reservoir from which increased amounts of acylation stimulating protein, a cleavage product of complement C3, could be produced.