Hepatitis C virus replication in mice with chimeric human livers.

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Mesenchymal stem cell therapy inhibited inflammatory and profibrotic pathways induced by partial bladder outlet obstruction and prevented high-pressure urine storage.

BACKGROUND: Partial bladder outlet obstruction (pBOO) is characterized by an initial inflammatory response that progresses to smooth muscle hypertrophy and fibrosis. Current treatment modalities carry high risk of morbidity. Mesenchymal stem cells (MSCs) are undifferentiated adult cells with reparative, immunomodulatory, and anti-inflammatory capacities. The ability of MSCs to inhibit inflammatory and profibrotic pathways in bladder cells has been recently reported. OBJECTIVES: This study aimed to investigate the therapeutic effects of MSCs on pBOO-induced inflammatory, profibrotic signaling pathways and end-organ physiology. MATERIALS AND METHODS: Twenty Sprague Dawley rats were randomly assigned to 5 groups: unobstructed controls, pBOO for 2 and 4 weeks, pBOO+MSCs for 2 and 4 weeks. Partial bladder outlet obstruction was surgically induced followed by intravenous injection of MSCs. Endpoint urodynamics was performed, and bladder tissue harvested for analysis. Reverse transcription real time polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to study gene and protein expression of major inflammatory and profibrotic markers. RESULTS: Partial bladder outlet obstruction resulted in an upregulation of transforming growth factor beta (TGFß1), mothers against decapentaplegic homolog 2/3 (SMAD2/3), hypoxia inducible factor 1 alpha (HIF1α), hypoxia inducible factor 3 alpha (HIF3α), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNFα), mechanistic target of rapamycin (mTOR), p70 ribosomal S6 protein kinase (p70 S6K), collagen 1 (COL1), and collagen 3 (COL3) expression in a time-dependent manner. This was coupled with a downregulation of interleukin (IL)-10 expression. Increase of bladder fibrosis was directly related to the duration of pBOO and associated with high urine storage pressure. Injected MSCs were identified in the bladder 4 weeks after therapy. The immunomodulatory effect of MSCs(defined by reduced TNFα and increased IL-10 and VEGF) was most predominant 2 weeks after therapy. Significant downregulation of profibrotic genes occurred 4 weeks after therapy. End filling pressure, hypertrophy, and fibrosis were significantly reduced after MSC therapy (P < 0.05). DISCUSSION: Mesenchymal stem cell therapy led to a profound systematic improvement of the obstructed bladder. This included an initial anti-inflammatory response and a subsequent antifibrotic reaction. Essentially, both phases were associated with a reduction of urine storage pressure. The intravenously injected MSCs were tracked in the bladder. However, their presence in non-target organs such as the lungs, spleen, and liver was not tracked. CONCLUSIONS: Partial bladder outlet obstruction induced significant upregulation of hypoxic, inflammatory, and profibrotic markers. Mesenchymal stem cell therapy potently inhibited these pathways and improved bladder function.

Acidosis and lipopolysaccharide from Escherichia coli B:055 cause hyperpermeability of rumen and colon tissues.

The objective of the present investigation was to evaluate the effects of acidic pH of the perfusate and presence of lipopolysaccharide (LPS) on permeability of rumen and colon mucosal tissues to mannitol and LPS using the Ussing chamber system. Rumen and colon tissues (n = 8), obtained from slaughtered feedlot steers, were tested for changes in permeability to (3)H-mannitol under pH of 4.5, 5.5, and 6.5 for rumen and at 5.5, 6.5, and 7.4 for colon, with or without LPS from Escherichia coli B:055 at 500 microg/mL. The (3)H-Mannitol was added at 10 microL (525.4 GBq/mmol) on the mucosal side of the Ussing chamber to detect changes in permeability, and 4 samples were taken at 20, 25, 30, and 35 min from the serosal side. Permeability of rumen and colon mucosa to (3)H-mannitol increased 6- and 5-fold, respectively, at acidic pH values of 4.5 and 5.5 and in the presence of 500 micro/mL of LPS. In contrast, LPS did not affect rumen and colon permeability at pH that ranged from 5.5 and 7.4. Translocation of LPS across the rumen and colon mucosa of cattle was not pH dependent. The LPS translocated through these tissues if present at the mucosal side. In conclusion, the permeability of rumen and colon tissues to (3)H-mannitol increased in presence of LPS and under acidic pH, whereas LPS permeated through mucosal tissues independently of the pH of the perfusate. Further research is warranted to understand the mechanism(s) by which acidic pH of the rumen digesta and presence of LPS make rumen and colon tissues "leaky".

Glycogen phosphorylase activity during the cold storage of liver: A limiting effect on glycolytic flux and energy production.

This study examined the effects of dibutyryl-cyclic adenosine monophosphate (db-cAMP) and okadaic acid (a specific inhibitor of protein phosphatases 1 and 2A) as additives to a cold storage solution. The effects on levels of glycogen phosphorylase, the resultant effects on flux through the glycolytic pathway, and the consequences of these changes on adenylate (ATP, ADP, and AMP) levels in rat liver during a 24-hr period of cold hypoxia were studied. The rapid transition to anaerobic metabolism was reflected in the increases in lactate levels for all groups. Total lactate accumulation in control livers (flushed and stored with a histidine-lactobionate-raffinose solution) was 9.8 micromol/g. The one notable difference between the control and experimental groups was the total lactate increase in one of the groups treated with db-cAMP; lactate accumulation was 16.0 micromol/g. There was a preferential maintenance of ATP that correlated with the increased flux through glycolysis observed with db-cAMP treatment; levels were 0.4-0.6 micromol/g higher than control group values between 2 and 10 hr of storage. In the control group, levels of glycogen phosphorylase in the active 'a' form began to decrease within 1 hr of exposure to cold hypoxic storage. Values dropped from 86% to 78% within the first 1 hr and by 10 hr, % 'a' was 57%. The separate addition of db-cAMP and okadaic acid resulted in a sustained maintenance of phosphorylase % 'a' throughout the entire cold hypoxic storage period; % 'a' values at 10 hr ranged from 75% to 81%. The major finding of this study was the clear and distinct correlation between phosphorylase % 'a' and total lactate accumulation (index of flux through glycolysis). This relationship was statistically significant after only 1 hr of storage, with a correlation coefficient of r=0.52 (P<0.025); however, the correlation became stronger as the time of storage progressed (by 10 hr, r=0.72; P<0.001). According to the relationship established, the maximum theoretical limit for lactate accumulation with 100% phosphorylase 'a' is approximately 30 micromol/g lactate. This finding suggests that glycogen phosphorylase and not necessarily glycogen content is one major determinant in maintaining anaerobic metabolism and energy production during cold liver storage. Hence, previous experiments that investigated the effects of nutritional status and glycogen content on tissue viability after experimental transplantation need to be reassessed.

Investigation of a primary requirement of organ preservation solutions: supplemental buffering agents improve hepatic energy production during cold storage.

BACKGROUND: This study was designed to investigate the effects of a modified University of Wisconsin (UW) solution supplemented with one of four buffering agents (histidine, bicine [N,N-bis(2-hydroxyethyl)glycine], tricine [N-tris(hydroxymethyl)methylglycine], and Tris) on liver metabolism during cold ischemic storage. METHODS: Rat livers were flushed and stored for a maximum period of 24 hr at 4 degrees C, and tissue energetics, substrate, and anaerobic end-products were assessed; the group exhibiting the best results during storage was recovered in a 60-min period of warm reperfusion. Relative buffering capacities of the experimental solutions (measured over physiological pH range, in mM H+/L) were: UW, 4.1; histidine+UW, 9.8; Tris+UW, 19.0; bicine+UW, 22.5; tricine+UW, 26.8. RESULTS: In the UW group, ATP levels dropped rapidly over the first 4 hr; 1.0 micromol/g (40% of initial) remained after 4 hr of storage. By 2 hr, ATP levels in bicine- and tricine-treated groups were 0.5 and 1.1 micromol/g greater than in the UW-stored livers and by 10 hr, ATP in bicine-treated livers was twofold that of the control (UW) group. Total adenylate levels also reflected a superior elevation of cellular energetics; even after 24 hr, quantities were 1.4 and 2.0 micromol/g higher than the UW group in bicine- and histidine-supplemented organs. The increase in energetics occurred as a result of increased flux through the major anaerobic energy-producing pathway, glycolysis. The glycolytic rate was significantly greater at storage times > 10 hr with solutions supplemented with bicine, histidine, and tricine. Final values for net lactate accumulation over the entire 24-hr storage period were: UW, 10.1 micromol/g; histidine, 14.3 micromol/g; bicine, 15.2 micromol/g; tricine, 13.8 micromol/g. Activities of glycogen phosphorylase revealed that the activity of this enzyme dropped by 50% within 2 hr of storage in UW. However, histidine and bicine supplementation resulted in a substantial elevation of phosphorylase "a" over 4 hr and 10 hr, respectively. The best buffer of the four examined in this study was bicine; energetics, glycolytic flux, and patterns of adenylate regeneration upon reperfusion were markedly superior to modified UW solution. CONCLUSION: The results of this study suggest that supplementing the "gold standard" UW solution with an additional buffering agent (in order of efficacy: bicine>tricine>histidine) may improve the metabolic status of livers during clinical organ retrieval/storage.

Novel approaches toward early diagnosis of islet allograft rejection.

BACKGROUND: The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation. METHODS: (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred. RESULTS: (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%. CONCLUSIONS: Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

Improvement of pancreatic islet isolation outcomes using glutamine perfusion during isolation procedure.

During procurement, isolation, and transplantation, islets are exposed to high levels of oxidative stress triggering a variety of signaling pathways that can ultimately lead to cell death. Glutamine is an important cellular fuel and an essential precursor for the antioxidant glutathione. The aim of this study was to examine the role of intraductal glutamine administration in facilitating recovery of isolated rat islets from pancreases subjected to a clinically relevant period of warm ischemia. Islets were isolated in Sprague-Dawley (SD) rats (n = 18 per group). Pancreata in groups 1 and 2 were procured immediately while groups 3 and 4 were subjected to 30-min warm ischemia. Groups 2 and 4 were treated intraductally with 5 mM glutamine prior to pancreatectomy. Exposure to 30-min warm ischemia significantly reduced islet yield [groups 1 & 2 (nonischemia): 503 +/- 29 islets/rat vs. groups 3 & 4 (ischemia): 247 +/- 26 islets/rat; p < 0.05]. Intraductal glutamine treatment significantly improved islet yield when pancreata were subjected to 30-min warm ischemia [144 +/- 16 islets/rat without glutamine (group 3) vs. 343 +/- 36 islets/rat with glutamine (group 4), p < 0.05]. Glutamine also significantly improved islet viability (values were 50 +/- 4% in group 4 vs. 27 +/- 3% in group 3, p < 0.05). Similarly, glutathione (reduced) levels were significantly elevated in both glutamine-treated groups; however, this increase was greatest in tissues exposed to ischemia (2.76 +/- 0.04 nmol/mg protein in group 4 vs. 1.66 +/- 0.04 nmol/mg protein in group 3, p < 0.05). Intraductal glutamine administration considerably improves the islet yield, viability, and augments endogenous glutathione levels in pancreata procured after a clinically relevant period of ischemia. Intraductal administration of glutamine at the time of digestive enzyme delivery into the harvested pancreas may represent a simple yet effective tool to improve islet yields in clinical isolations.

Resuscitation of the ischemically damaged human pancreas by the two-layer method prior to islet isolation.

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata. This study determined the effect of additional preservation of ischemically damaged human pancreata by the TLM before islet isolation. Human pancreata were procured from cadaveric organ donors and preserved by the TLM for 3.2 +/- 0.5 hours (mean +/- SEM) at 4 degrees C after 11.1 +/- 0.9 hours of cold storage in University of Wisconsin solution (UW) (TLM group), or by cold UW alone for 11.0 +/- 0.3 hours (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro function of isolated islets were significantly increased in the TLM group compared with the UW group. In the metabolic assessment of human pancreata, ATP levels were significantly increased after the TLM preservation. This study showed that additional short-term preservation by the TLM resuscitates the viability of ischemically damaged human pancreata before islet isolation, leading to improvements in islet recovery and in vitro function of isolated islets.

VSL#3 probiotics provide protection against acute intestinal ischaemia/reperfusion injury.

Acute intestinal ischaemia/reperfusion injury (AII/R) is an adaptive physiologic response during critical illness, involving mesenteric vasoconstriction and hypoperfusion. Prevention of AII/R in high risk patient populations would have a significant impact on morbidity and mortality. The purpose of this study was to investigate the protective effects of VSL#3 probiotic treatment in a murine model of AII/R. Adult 129/SvEv mice were subjected to an experimental AII/R model using superior mesenteric artery occlusion. Animals were pre-treated with either three days or two weeks of VSL#3 probiotics. Local tissue injury markers were assessed by levels of myeloperoxidase and activation of nuclear factor kappa B (NFкB). Systemic and local cytokines, including interleukin (IL)-1ß, IL- 10, TNFα, and interferon gamma were measured by ELISA and multiplex fluorescent detection. VSL#3 probiotics reduced local tissue inflammation and injury due to AII/R. A two-week course of VSL#3 was more effective than a shorter three-day course. The reduction in local inflammation from the two-week course of VSL#3 is correlated to a significant reduction in levels of active IL-1ß, and tissue levels of myeloperoxidase. Levels of active NFкB were significantly elevated in the vehicle-fed AII/R mice, corroborating with tissue inflammation, which were attenuated by VSL#3 administrations. VSL#3 did not cause any systemic inflammation or lung injury. VSL#3 probiotics are effective in reducing local tissue injury from AII/R by down-regulating pro-inflammatory mediators and immune cell recruitment. This study highlights a potential role for VSL#3 in management of patients at high risk for AII/R.

Improved outcomes in islet isolation and transplantation by the use of a novel hemoglobin-based O2 carrier.

During isolation, islets are exposed to warm ischemia. In this study, intraductal administration of oxygenated polymerized, stroma-free hemoglobin-pyridoxalated (Poly SFH-P) was performed to improve O2 delivery. Rat pancreata subjected to 30-min warm ischemia were perfused intraductally with collagenase in oxygenated Poly SFH-P/RPMI or RPMI (control). PO2 was increased by Poly SFH-P (381.7 +/- 35.3 mmHg vs. 202.3 +/- 28.2, p = 0.01) and pH maintained within physiological range (7.4-7.2 vs. 7.1-6.6, p = 0.009). Islet viability (77% +/- 4.6 vs. 63% +/- 4.7, p = 0.04) was improved and apoptosis lower with Poly SFH-P (caspase-3: 34,714 +/- 2167 vs. 45,985 +/- 1382, respectively, p = 0.01). Poly SFH-P improved islet responsiveness to glucose as determined by increased intracellular Ca2+ levels and improved insulin secretion (SI 5.4 +/- 0.1 vs. 3.1 +/- 0.2, p = 0.03). Mitochondrial integrity was improved in Poly SFH-P-treated islets, which showed higher percentage change in membrane potential after glucose stimulation (14.7% +/- 1.8 vs. 9.8 +/- 1.4, respectively, p < 0.05). O2 delivery by Poly SFH-P did not increase oxidative stress (GSH 7.1 +/- 2.9 nm/mg protein for Poly SFH-P vs. 6.8 +/- 2.4 control, p = 0.9) or oxidative injury (MDA 1.8 +/- 0.9 nmol/mg protein vs. 6.2 +/- 2.4, p = 0.19). Time to reach normoglycemia in transplanted diabetic nude mice was shorter (1.8 +/- 0.4 vs. 7 +/- 2.5 days, p = 0.02), and glucose tolerance improved in the Poly SFH-P group (AUC 8106 +/- 590 vs. 10,863 +/- 946, p = 0.03). Oxygenated Poly SFH-P improves islet isolation and transplantation outcomes by preserving mitochondrial integrity.

Metabolic effects of dehydration on an aquatic frog, Rana pipiens.

Cellular responses to dehydration were analyzed in six organs of leopard frogs Rana pipiens. Frogs at 5 degrees C endured the loss of up to 50% of their total body water content but water contents of individual organs were strongly defended. Skeletal muscle water content was strongly affected by dehydration, dropping from 80.7% of wet mass in controls to 67.2% in frogs that had lost 50% of their total body water. However, water contents of internal organs dropped by only 3-8% of their wet masses. Water contents of all organs except skeletal muscle were fully restored by 24h of rehydration in water at 5 degrees C. Dehydration had no consistent effect on the protein content of five organs but in a sixth, the kidney, protein levels were elevated (by 60-72%) at the higher levels of dehydration and during rehydration. Dehydration led to a rapid increase in glucose concentration in the liver; compared with control values of 13 +/- 2 nmol mg-1 protein, levels were doubled by 12.2% dehydration and continued to increase to a maximum of 307 +/- 44 nmol mg-1 protein (20 mumol g-1 wet mass) in 50% dehydrated frogs. Glucose accumulation was supported by a decrease in liver glycogen content and a parallel rise in glucose 6-phosphate levels, but not in the levels of other glycolytic intermediates, confirming that glycogenolytic flux was being directed into glucose synthesis. Blood glucose levels also increased as a function of increasing dehydration, reaching values 13.8 times higher than controls, but only the kidney and brain showed a significant accumulation of glucose over the course of dehydration.(ABSTRACT TRUNCATED AT 250 WORDS)

Dehydration tolerance in wood frogs: a new perspective on development of amphibian freeze tolerance.

Wood frogs, Rana sylvatica, tolerate the loss of 50-60% of total body water during experimental dehydration. The rate of water loss for unprotected frogs is the same whether animals are frozen (at -2 degrees C) or unfrozen (at 1 degrees C) but is greatly reduced when frogs are frozen under a protective layer of moss. Dehydrational death could occur in as little as 7-9 days for unprotected animals; this indicates the importance for winter survival of selecting well-protected and damp hibernation sites. Prior dehydration affected the cooling and freezing properties of frogs, reducing supercooling point and the amount of ice formed after 24 h at -2 degrees C and acting synergistically with freezing exposure in stimulating cryoprotectant synthesis. Analysis of the effects of controlled dehydration at 5 degrees C showed that changes in body water content alone (without freezing) stimulated liver glycogenolysis and the export of high concentrations of glucose into blood and other organs. Autumn-collected frogs dehydrated to 50% of total body water lost showed glucose levels of 165-1,409 nmol/mg protein in different organs, increases of 9- to 313-fold compared with control values and reaching final levels very similar to those induced by freezing exposure. The data support the proposal that various adaptations for natural freeze tolerance may have been derived from preexisting mechanisms for dealing with water stress in amphibians and that cell volume change may be one of the signals involved in triggering and sustaining molecular adaptations (e.g., cryoprotectant output) that support freezing survival.

Natural freezing survival by painted turtles Chrysemys picta marginata and C. picta bellii.

Hatchlings of both the Midland (Chrysemys picta marginata) and Western (C. picta bellii) subspecies of the painted turtle tolerate the freezing of extracellular body fluids while overwintering in terrestrial nests. Fall-collected hatchlings survived 3 days of continuous freezing at -2.5 degrees C, with ice contents of 43.5 +/- 1.0% of total body water (SE; n = 24) for C. picta marginata and 46.5 +/- 0.8% (n = 32) for C. picta bellii. Survival times dropped to 4-5 h when temperature was lowered to -4 degrees C, correlated with ice contents of greater than or equal to 50%. However, C. picta marginata tested immediately after excavation from nests in the spring showed greater freeze tolerance, with survival extending to 11 days at -2.5 degrees C and a higher mean ice content of 50.2 +/- 1.2% (n = 6). Spring hatchlings also had high supercooling points, -1.07 +/- 0.13 degrees C (n = 8), that dropped within 3 days to -4.83 +/- 0.83 degrees C (n = 4), suggesting a breakdown of endogenous ice-nucleating agents when hibernation ended. A search for possible cryoprotectants showed that both subspecies accumulated glucose and lactate in liver during freezing (net increase = 3-13 mumols/g wet wt); both also maintained large free amino acid pools in organs, with taurine making up 21-47% of the total.

Responses to freezing exposure of hatchling turtles Trachemys scripta elegans: factors influencing the development of freeze tolerance by reptiles.

Hatchling red-eared turtles Trachemys (= Pseudemys) scripta elegans (Wied) from a Louisiana population display a significant ability to withstand the freezing of extracellular body fluids. All animals survived at least 2 h of freezing at -2.5 or -4 degrees C. At -2.5 degrees C, survival declined to 50% after 6 h of freezing and no animals recovered after 24 h or longer, when mean ice content reached 54.7 +/- 1.4% of total body water. At -4 degrees C, all turtles recovered from 4 h of freezing exposure with a mean ice content of 49.6 +/- 2.4%, but survival dropped sharply thereafter with no animals recovering after 8 h, when ice content had reached 64.5 +/- 0.7%. Survival times were substantially shorter and percentage ice values greater than comparable values for hatchling painted turtles (Chrysemys picta (Schneider)) from northern populations subjected to identical freezing exposures. The ability to synthesize cryoprotectants in response to freezing was poorly developed in T. s. elegans; maximal accumulation of glucose was only 3.2 mumol g-1 wet mass in liver. Lactate content increased two- to threefold in oxygen-sensitive organs (heart and brain) during freezing, but levels of lactate and other putative cryoprotectants were unchanged in other organs. Total free amino acid content rose significantly in liver, muscle and blood during freezing; increased taurine concentration was primarily responsible for the changes in liver and blood. The capacity for freezing survival by T. s. elegans hatchlings from southern populations would be of limited use for hibernation in a cold climate, but the metabolic responses to freezing displayed by these animals might be enhanced by northern populations to increase their freeze tolerance.

Effects of dehydration on organ metabolism in the frog Pseudacris crucifer: hyperglycemic responses to dehydration mimic freezing-induced cryoprotectant production.

The metabolic effects of evaporative water loss at 5 degrees C were assessed for both fall- and spring-collected spring peepers Pseudacris crucifer. Frogs readily endured the loss of 50% of total body water. During dehydration organ water content was defined with no change in water content in skeletal muscle, gut, and kidney of 50% dehydrated frogs and reduced water content in liver, brain and heart. Dehydration stimulated a rapid and massive increase in liver glucose production. In fall-collected frogs liver glucose rose by 120-fold to 2690 +/- 400 nmol.mg protein-1 or 220 mumol.g ww-1 in 50% dehydrated frogs and glucose in other organs increased by 2.6- to 60-fold. Spring-collected frogs showed the same qualitative response to dehydration although absolute glucose levels were lower, rising maximally by 8.4-fold in liver. Glucose synthesis was supported by glycogenolysis in liver and changes in the levels of glycolytic intermediates in liver indicated that an inhibitory block at the phosphofructokinase locus during desiccation helped to divert hexose phosphates into the production of glucose. Liver energy status (ATP, total adenylates, energy charge) was maintained even after the loss of 35% of total body water but at 50% dehydration all parameters showed a sharp decline; for example, energy charge fell from about 0.85 to 0.42. Severe dehydration also led to an accumulation of lactate in four organs, probably hypoxia-induced due to impaired circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of glycolysis by histidine buffers in mammalian liver during cold hypoxia.

This study was designed to address the reasons why glycolysis in mammalian liver is unable to function more efficiently during periods of cold hypoxia. Our hypothesis was that control of intracellular pH, by use of amino acid buffers with high pKa values, would allow prolonged flux through glycolysis and better maintenance of liver high-energy adenine nucleotide pool. The effects of two concentrations of histidine (90 and 180 mM) and one of carnosine (90 mM), a histidyl dipeptide, on energy metabolism and levels of glycolytic substrate (glucose) and anaerobic endproduct (lactate) were investigated during cold hypoxia using rat livers to model the mammalian system. The transition to anaerobic metabolism was apparent by an immediate rise in lactate levels upon entry into cold hypoxia. By 10-14 h hypoxia, contents of the endproduct had increased by 10, 13.5, and 14.5 mumol/g in buffers containing 90 and 180 mM histidine and 90 mM carnosine, respectively. As well, ATP, total adenylate contents, and "energy charge" ratios exhibited a rapid decline from initial values of 2.3-3.3 mumol/g, 4.3-5.5 mumol/g, and 0.64-0.75, respectively, over the first 2-4 h of cold hypoxia. With respect to efficacy, the 180 mM histidine buffer exhibited the most positive maintenance of adenylate levels, followed closely by 90 mM carnosine, and finally 90 mM histidine as the least effective of the three buffers. Nevertheless, all three buffers examined in this study showed positive effects compared to similarly treated livers stored in a solution of minor buffering capacity (a citrate-based solution) over the same time period. The data support the hypothesis that glycolytic flux and cellular energetics can be maintained by the inclusion of efficient buffering agents during periods of cold hypoxia.