The degradation of semisynthetic tritiated insulin by perfused mouse livers.
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ABSTRACT:
SUBMITTER: Halban PA
PROVIDER: S-EPMC1164249 | biostudies-other | 1976 Nov
REPOSITORIES: biostudies-other
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Project description:Isolated liver perfusion systems have been used to characterize intrinsic metabolic changes in liver as a result of various perturbations, including systemic injury, hepatotoxin exposure, and warm ischemia. Most of these studies were done using hyperoxic conditions (95% O(2)) but without the use of oxygen carriers in the perfusate. Prior literature data do not clearly establish the impact of oxygenation, and in particular that of adding oxygen carriers to the perfusate, on the metabolic functions of the liver. Therefore, herein the effects of oxygen delivery in the perfusion system on liver metabolism were investigated by comparing three modes of oxygenation. Rat livers were perfused via the portal and hepatic veins at a constant flow rate of 3 mL/min/g liver in a recirculating perfusion system. In the first group, the perfusate was equilibrated in a membrane oxygenator with room air (21% O(2)) before entering the liver. In the second group, the perfusate was equilibrated with a 95% O(2)/5% CO(2) gas mixture. In the third group, the perfusate was supplemented with washed bovine red blood cells (RBCs) at 10% hematocrit and also equilibrated with the 95% O(2)/5% CO(2) gas mixture. Oxygen and CO(2) gradients across the liver were measured periodically with a blood gas analyzer. The rate of change in the concentration of major metabolites in the perfusate was measured over time. Net extracellular fluxes were calculated from these measurements and applied to a stoichiometric-based optimization problem to determine the intracellular fluxes and active pathways in the perfused livers. Livers perfused with RBCs consumed oxygen at twice the rate observed using hyperoxic (95% O(2)) perfusate without RBCs, and also produced more urea and ketone bodies. At the flow rate used, the oxygen supply in perfusate without RBCs was just sufficient to meet the average oxygen demand of the liver but would be insufficient if it increased above baseline, as is often the case in response to environmental perturbations. Metabolic pathway analysis suggests that significant anaerobic glycolysis occurred in the absence of RBCs even using hyperoxic perfusate. Conversely, when RBCs were used, glucose production from lactate and glutamate, as well as pathways related to energy metabolism were upregulated. RBCs also reversed an increase in PPP fluxes induced by the use of hyperoxic perfusate alone. In conclusion, the use of oxygen carriers is required to investigate the effect of various perturbations on liver metabolism.
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Project description:1. This paper describes the semisynthesis of [3H]insulin of high specific radioactivity which is indistinguishable from native insulin in its hypoglycaemic activity towards mice. 2. The maximum specific radioactivity so far obtained is 20 Ci/mmol. At this value the recommended scale of preparation is between 1 mCi (30 mug of product) and 10mCi (300 mug of product). Reaction volumes are then of the order of 10 mul. 3. Special problems arise when the techniques of peptide synthesis are applied to the production of such small quantities of material. We describe these problems and discuss the means of overcoming them.
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Project description:(1) We [Muir, Offord & Davies (1986) Biochem. J. 237, 631-637 and Davies, Muir & Offord (1986) Biochem. J. 240, 609-612] have previously identified a major product in the degradation of insulin by insulin proteinase (the N-terminal fragment produced by cleavage between residues LeuA13 and TyrA14, SerB9 and HisB10) together with evidence for a minor cleavage site between HisB10 and LeuB11 or between LeuB11 and ValB12. (2) We now present evidence for minor sites of cleavage between TyrA14 and GlnA15, GluB13 and AlaB14 as well as HisB10 and LeuB11.
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Project description:AIM: Transformation and possible metabolic effects of extracellular NAD+ were investigated in the livers of mice (Mus musculus; Swiss strain) and rats (Rattus novergicus; Holtzman and Wistar strains). METHODS: The livers were perfused in an open system using oxygen-saturated Krebs/Henseleit-bicarbonate buffer (pH 7.4) as the perfusion fluid. The transformation of NAD+ was monitored using high-performance liquid chromatography. RESULTS: In the mouse liver, the single-pass metabolism of 100 micromol/L NAD+ was almost complete; ADP-ribose and nicotinamide were the main products in the outflowing perfusate. In the livers of both Holtzman and Wistar rats, the main transformation products were ADP-ribose, uric acid and nicotinamide; significant amounts of inosine and AMP were also identified. On a weight basis, the transformation of NAD+ was more efficient in the mouse liver. In the rat liver, 100 micromol/L NAD+ transiently inhibited gluconeogenesis and oxygen uptake. Inhibition was followed by a transient stimulation. Inhibition was more pronounced in the Wistar strain and stimulation was more pronounced in the Holtzman strain. In the mouse liver, no clear effects on gluconeogenesis and oxygen uptake were found even at 500 micromol/L NAD+. CONCLUSION: It can be concluded that the functions of extracellular NAD+ are species-dependent and that observations in one species are strictly valid for that species. Interspecies extrapolations should thus be made very carefully. Actually, even variants of the same species can demonstrate considerably different responses.
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Project description:1. In livers from fed rats perfused with homologous whole blood of a haematocrit value of 37%, insulin decreased the perfusate concentrations of glucose and amino acids, production of ketone bodies (3-hydroxybutyrate + acetoacetate) and increased bile flow. 2. Perfusion with blood diluted with buffer to a haematocrit value of 17% decreased hepatic O2 consumption by 40-50%. Perfusate concentrations of glucose and lactate, the rate of ketogenesis and the ratios [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] were all increased. 3. In livers perfused with blood of diminished haematocrit, effects of insulin on perfusate glucose an amino acids, ketogenesis and bile flow were abolished.
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Project description:The Langendorff-perfused model is a powerful tool to study biological responses in the isolated heart in the absence of confounders. The model has been adapted recently to enable study of the isolated mouse heart and the effects of genetic manipulation. Unfortunately, the small size and fragility of the mouse heart pose significant challenges, limiting application of the Langendorff model to the study of adult mice. Cardiac development is a complex and dynamic process that is incompletely understood. Thus, establishing an isolated-perfused heart model in the newborn mouse would be an important and necessary advance. Here we present a method to successfully cannulate and perfuse the isolated newborn murine heart. We describe the basic and fundamental physiological characteristics of the ex-vivo retrograde-perfused beating neonatal heart in wild-type C57Bl/6 male mice. Our approach will enable future study of the physiological and pharmacological responses of the isolated immature murine heart to enhance knowledge of how developmental cardiac biology impacts health and disease.•The Langendorff model is a powerful tool to study the heart without confounders.•An isolated-perfused newborn murine heart model has yet to be established.•We demonstrate the first successful isolated neonatal murine heart preparation.
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Project description:Major depressive disorder (MDD) is a common mood disorder. Gut microbiota may be involved in the pathogenesis of depression via the microbe-gut-brain axis. Liver is vulnerable to exposure of bacterial products translocated from the gut via the portal vein and may be involved in the axis. In this study, germ-free mice underwent fecal microbiota transplantation from MDD patients and healthy controls. Behavioral tests verified the depression model. Metabolomics using gas chromatography-mass spectrometry, nuclear magnetic resonance, and liquid chromatography-mass spectrometry determined the influence of microbes on liver metabolism. With multivariate statistical analysis, 191 metabolites were distinguishable in MDD mice from control (CON) mice. Compared with CON mice, MDD mice showed lower levels for 106 metabolites and higher levels for 85 metabolites. These metabolites are associated with lipid and energy metabolism and oxidative stress. Combined analyses of significantly changed proteins in livers from another depression model induced by chronic unpredictive mild stress returned a high score for the Lipid Metabolism, Free Radical Scavenging, and Molecule Transports network, and canonical pathways were involved in energy metabolism and tryptophan degradation. The two mouse models of depression suggest that changes in liver metabolism might be involved in the pathogenesis of MDD. Conjoint analyses of fecal, serum, liver, and hippocampal metabolites from fecal microbiota transplantation mice suggested that aminoacyl-tRNA biosynthesis significantly changed and fecal metabolites showed a close relationship with the liver. These findings may help determine the biological mechanisms of depression and provide evidence about "depression microbes" impacting on liver metabolism.
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Project description:AIMS/HYPOTHESIS:Podocytes are insulin-responsive cells of the glomerular filtration barrier and are key in preventing albuminuria, a hallmark feature of diabetic nephropathy. While there is evidence that a loss of insulin signalling to podocytes is detrimental, the molecular mechanisms underpinning the development of podocyte insulin resistance in diabetes remain unclear. Thus, we aimed to further investigate podocyte insulin responses early in the context of diabetic nephropathy. METHODS:Conditionally immortalised human and mouse podocyte cell lines and glomeruli isolated from db/db DBA/2J mice were studied. Podocyte insulin responses were investigated with western blotting, cellular glucose uptake assays and automated fluorescent imaging of the actin cytoskeleton. Quantitative (q)RT-PCR was employed to investigate changes in mRNA. Human cell lines stably overproducing the insulin receptor (IR) and nephrin were also generated, using lentiviral constructs. RESULTS:Podocytes exposed to a diabetic environment (high glucose, high insulin and the proinflammatory cytokines TNF-? and IL-6) become insulin resistant with respect to glucose uptake and activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling. These podocytes lose expression of the IR as a direct consequence of prolonged exposure to high insulin concentrations, which causes an increase in IR protein degradation via a proteasome-dependent and bafilomycin-sensitive pathway. Reintroducing the IR into insulin-resistant human podocytes rescues upstream phosphorylation events, but not glucose uptake. Stable expression of nephrin is also required for the insulin-stimulated glucose uptake response in podocytes and for efficient insulin-stimulated remodelling of the actin cytoskeleton. CONCLUSIONS/INTERPRETATION:Together, these results suggest that IR degradation, caused by high levels of insulin, drives early podocyte insulin resistance, and that both the IR and nephrin are required for full insulin sensitivity of this cell. This could be highly relevant for the development of nephropathy in individuals with type 2 diabetes, who are commonly hyperinsulinaemic in the early phases of their disease.
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