Separation of enzymatically distinct populations of trout hepatocytes

1991 ◽  
Vol 69 (2) ◽  
pp. 420-426 ◽  
Author(s):  
Thomas P. Mommsen ◽  
Eva Danulat ◽  
M. Emilia Gavioli ◽  
Glen D. Foster ◽  
Thomas W. Moon
Keyword(s):  
Hepatic Vein ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Citrate Synthase ◽  
Short Pulse ◽  
Gradient Centrifugation ◽  
Cellular Heterogeneity ◽  
Relative Distribution ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Collagenase Perfusion ◽  
Glucagon Like Peptide

Two different approaches were chosen to investigate the heterogeneity of rainbow trout (Oncorhynchus mykiss) hepatocytes. (i) Hepatocytes were isolated following selective liver infusion with a short pulse of digitonin from either the portal or the hepatic vein. Cell isolates, presumably enriched in cells from either "periportal" or "perivenous" liver regions, were then compared with cells obtained from the nondigitonized part of the same liver (control). No significant differences between hepatocytes were found in the activities of six key metabolic enzymes or in the rate of pyruvate oxidation. All cell isolates were responsive to glucagon, glucagon-like peptide, and insulin (all at 10 nM). If the liver was digitonized through the hepatic vein, resulting cell isolates revealed higher rates of gluconeogenesis from pyruvate, and gluconeogenic flux was more responsive to glucagon (at 10 nM) than controls. The experimental evidence for metabolic zonation in trout liver is weak. (ii) Enzymatically different subpopulations of hepatocytes were successfully separated by Percoll gradient centrifugation after isolation of cells by the conventional collagenase perfusion technique. Two prominent subpopulations of hepatocytes differed in the activities of glycogen phosphorylase, pyruvate kinase, lactate dehydrogenase, citrate synthase, glutamate dehydrogenase, phosphoenolpyruvate carboxykinase, and alanine and aspartate aminotransferases, but not in their content of glycogen. However, the relative distribution of enzymes did not follow the mammalian pattern of perivenous or periportal hepatocytes. These data support cellular heterogeneity in the trout liver, with gluconeogenesis and oxidative metabolism dominating in some hepatocytes, whereas others display a relatively larger scope for glycogen breakdown, glycolysis, and amino acid metabolism.

scholarly journals The impact of obesity, sex, and diet on hepatic glucose production in cats

2009 ◽  
Vol 296 (4) ◽  
pp. R936-R943 ◽  
Author(s):  
Saskia Kley ◽  
Margarethe Hoenig ◽  
John Glushka ◽  
Eunsook S. Jin ◽  
Shawn C. Burgess ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Citrate Synthase ◽  
Hepatic Glucose Production ◽  
Glucose Production ◽  
Endogenous Glucose Production ◽  
Hepatic Glucose Metabolism ◽  
Peripheral Insulin Resistance ◽  
Pyruvate Cycling ◽  
Hepatic Glucose ◽  
The Impact

Obesity is a risk factor for type 2 diabetes in cats. The risk of developing diabetes is severalfold greater for male cats than for females, even after having been neutered early in life. The purpose of this study was to investigate the role of different metabolic pathways in the regulation of endogenous glucose production (EGP) during the fasted state considering these risk factors. A triple tracer protocol using 2H2O, [U-13C3]propionate, and [3,4-13C2]glucose was applied in overnight-fasted cats (12 lean and 12 obese; equal sex distribution) fed three different diets. Compared with lean cats, obese cats had higher insulin ( P < 0.001) but similar blood glucose concentrations. EGP was lower in obese cats ( P < 0.001) due to lower glycogenolysis and gluconeogenesis (GNG; P < 0.03). Insulin, body mass index, and girth correlated negatively with EGP ( P < 0.003). Female obese cats had ∼1.5 times higher fluxes through phosphoenolpyruvate carboxykinase ( P < 0.02) and citrate synthase ( P < 0.05) than male obese cats. However, GNG was not higher because pyruvate cycling was increased 1.5-fold ( P < 0.03). These results support the notion that fasted obese cats have lower hepatic EGP compared with lean cats and are still capable of maintaining fasting euglycemia, despite the well-documented existence of peripheral insulin resistance in obese cats. Our data further suggest that sex-related differences exist in the regulation of hepatic glucose metabolism in obese cats, suggesting that pyruvate cycling acts as a controlling mechanism to modulate EGP. Increased pyruvate cycling could therefore be an important factor in modulating the diabetes risk in female cats.

Inter-tissue differences in mitochondrial enzyme activity, RNA and DNA in rainbow trout (Oncorhynchus mykiss)

1998 ◽  
Vol 201 (24) ◽  
pp. 3377-3384 ◽  
Author(s):  
S. C. Leary ◽  
B. J. Battersby ◽  
C. D. Moyes
Keyword(s):  
Enzyme Activity ◽  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Citrate Synthase ◽  
Mitochondrial Protein ◽  
Molecular Organization ◽  
Mitochondrial Rna ◽  
Mitochondrial Enzyme ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
The Relationship

We examined whether the relationships between mitochondrial enzyme activity, mitochondrial DNA (mtDNA) and mitochondrial RNA (mtRNA) were conserved in rainbow trout (Oncorhynchus mykiss) tissues that differ widely in their metabolic and molecular organization. The activity of citrate synthase (CS), expressed either per gram of tissue or per milligram of total DNA, indicated that these tissues (blood, brain, kidney, liver,cardiac, red and white muscles) varied more than 100-fold in mitochondrial content. Several-fold differences in the levels of CS mRNA per milligram of DNA and CS activity per CS mRNA were also observed, suggesting that fundamental differences exist in the regulation of CS levels across tissues. Although tissues varied 14-fold in RNA g-1, poly(A+) RNA (mRNA)was approximately 2 % of total RNA in all tissues. DNA g-1 also varied 14-fold across tissues, but RNA:DNA ratios varied only 2.5-fold. The relationship between two mitochondrial mRNA species (COX I, ATPase VI) and one mitochondrial rRNA (16S) species was constant across tissues. The ratio of mtRNA to mtDNA was also preserved across most tissues; red and white muscle had 10- to 20-fold lower levels of mtDNA g-1 but 7- to 10-fold higher mtRNA:mtDNA ratios, respectively. Collectively, these data suggest that the relationship between mitochondrial parameters is highly conserved across most tissues, but that skeletal muscles differ in a number of important aspects of respiratory gene expression ('respiratory genes'include genes located on mtDNA and genes located in the nucleus that encode mitochondrial protein) and mtDNA transcriptional regulation.

Incomplete Glyoxysomes Appearing at a Late Stage of Maturation of Cucumber Seeds

1979 ◽  
Vol 34 (12) ◽  
pp. 1232-1236 ◽  
Author(s):  
Wolfram Koller ◽  
Jürgen Frevert ◽  
Helmut Kindi
Keyword(s):  
Late Stage ◽  
Citrate Synthase ◽  
De Novo ◽  
Density Gradient Centrifugation ◽  
Isocitrate Lyase ◽  
Gradient Centrifugation ◽  
Protein Body ◽  
Electrophoretic Analysis ◽  
Malate Synthase ◽  
Lyase Activity

Seeds of cucumber fruits at a late stage of ripening were analyzed for microbodies and micro­body components. On isopycnic density gradient centrifugation of homogenates in the presence of EDTA, several particulate fractions were obtained: a light membraneous fraction (density d = 1.09-1.11 kg × 1-1), a mitochondria-enriched fraction (d = 1.21 kg × 1-1), a microbody-enriched fraction (d = 1.23 kg × 1-1), and a protein body fraction (d= 1.26 - 1.29 kg × 1-1). Microbo­dies were revealed by exactly coinciding peaks of malate synthase, catalase and crotonase; small proportions of citrate synthase and malate dehydrogenase were also present in this zone. Isocitrate lyase activity, however, did not occur in the seeds at this stage. The examination of enzyme activi­ties indicated the presence of microbodies which cannot function as competent glyoxysomes be­cause of the lack of isocitrate lyase. Moreover, de novo synthesis from [3H] leucine could be de­monstrated for malate synthase by means of immunoprecipitation of newly synthesized malate synthase and subsequent electrophoretic analysis.

Thermal acclimation in Antarctic fish: transcriptomic profiling of metabolic pathways

2011 ◽  
Vol 301 (5) ◽  
pp. R1453-R1466 ◽  
Author(s):  
Heidrun Sigrid Windisch ◽  
Raphaela Kathöver ◽  
Hans-Otto Pörtner ◽  
Stephan Frickenhaus ◽  
Magnus Lucassen
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Citrate Synthase ◽  
Thermal Sensitivity ◽  
Antarctic Fish ◽  
Control Group ◽  
Acclimation Temperature ◽  
Peroxisome Proliferator ◽  
Warm Acclimation ◽  
Peroxisome Proliferator Activated Receptors ◽  
Large Rearrangements

It is widely accepted that adaptation to the extreme cold has evolved at the expense of high thermal sensitivity. However, recent studies have demonstrated significant capacities for warm acclimation in Antarctic fishes. Here, we report on hepatic metabolic reorganization and its putative molecular background in the Antarctic eelpout ( Pachycara brachycephalum ) during warm acclimation to 5°C over 6 wk. Elevated capacities of cytochrome c oxidase suggest the use of warm acclimation pathways different from those in temperate fish. The capacity of this enzyme rose by 90%, while citrate synthase (CS) activity fell by 20% from the very beginning. The capacity of lipid oxidation by hydroxyacyl-CoA dehydrogenase remained constant, whereas phosphoenolpyruvate carboxykinase as a marker for gluconeogenesis displayed 40% higher activities. These capacities in relation to CS indicate a metabolic shift from lipid to carbohydrate metabolism. The finding was supported by large rearrangements of the related transcriptome, both functional genes and potential transcription factors. A multivariate analysis (canonical correspondence analyses) of various transcripts subdivided the incubated animals in three groups, one control group and two responding on short and long timescales, respectively. A strong dichotomy in the expression of peroxisome proliferator-activated receptors-1α and -β receptors was most striking and has not previously been reported. Altogether, we identified a molecular network, which responds sensitively to warming beyond the realized ecological niche. The shift from lipid to carbohydrate stores and usage may support warm hardiness, as the latter sustain anaerobic metabolism and may prepare for hypoxemic conditions that would develop upon warming beyond the present acclimation temperature.

scholarly journals Partial isolation and translation in vitro of messenger ribonucleic acid for phosphoenolpyruvate carboxykinase (guanosine triphosphate)

1976 ◽  
Vol 156 (3) ◽  
pp. 619-626 ◽  
Author(s):  
S M Tilghman ◽  
L M Fisher ◽  
L Reshef ◽  
F J Ballard ◽  
R W Hanson
Keyword(s):  
Gel Electrophoresis ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Messenger Ribonucleic Acid ◽  
Partial Isolation ◽  
Heterologous Cell ◽  
Rabbit Reticulocytes

1. mRNA was extracted from the livers of starved rats and incubated in a heterologous cell-free protein-synthesizing system derived from rabbit reticulocytes. The presence of newly synthesized phosphoenolpyruvate carboxykinase (GTP) was detected by immunoprecipitation with a specific antibody to the enzyme and analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The synthesis of the enzyme was dependent on the addition of rat liver RNA, whereas RNA isolated from rat spleen was inactive. If ovalbumin and anti-ovalbumin were used to form the immunoprecipitates, no radioactivity that migrated as phosphoenolpyruvate carboxykinase was detected. 3. The optimal concentrations of magnesium acetate and KCl for phosphoenolpyruvate carboxykinase synthesis were determined. 4. When polyribosomal RNA was separated by sucrose-gradient centrifugation, phosphoenolpyruvate carboxykinase mRNA migrated between 20 and 26 S in keeping with the high mol. wt. of the protein (85 000). 5. The presence of poly(A) in phosphoenolpyruvate carboxykinase mRNA was suggested by retention of mRNA activity on oligo(dT)-cellulose columns. 6. It was concluded that the cell-free synthesis of phosphoenolpyruvate carboxykinase can serve as a bioassay for intracellular phosphoenolpyruvate carboxykinase mRNA.

scholarly journals Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity

2004 ◽  
Vol 381 (2) ◽  
pp. 379-387 ◽  
Author(s):  
Tirumala Kumar CHOWDARY ◽  
Bakthisaran RAMAN ◽  
Tangirala RAMAKRISHNA ◽  
Chintalagiri Mohan RAO
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Quaternary Structure ◽  
Citrate Synthase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Nucleotide Sequence Analysis ◽  
Heat Shock Factor 1 ◽  
Induced Aggregation

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic α-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.

Enzyme activities in the Atlantic hagfish, Myxine glutinosa: changes with captivity and food deprivation

1986 ◽  
Vol 64 (5) ◽  
pp. 1080-1085 ◽  
Author(s):  
Glen D. Foster ◽  
T. W. Moon
Keyword(s):  
Food Deprivation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Citrate Synthase ◽  
Krebs Cycle ◽  
Metabolic Potential ◽  
Metabolizing Enzymes ◽  
Myxine Glutinosa ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Krebs Cycle Enzymes ◽  
Glucose 6 Phosphate Dehydrogenase

Selected Krebs cycle enzymes and carbohydrate, amino acid, and lipid metabolizing enzymes were assayed in the muscle and liver of newly captured (April, 4 °C; August, 15 °C), fed (for 7 months), and food-deprived (for 7 and 11 months) hagfish, Myxine glutinosa. Seasonal differences were found in the glycogen content of the muscle and liver of newly captured hagfish (lower in the cold temperature), while consistently high levels were maintained in the fed group. Food deprivation decreased the content. All enzymes measured were found in both tissues, except glucose-6-phosphate dehydrogenase (liver only) and glycerol kinase (absent in both tissues). Activities of the enzymes were lower than teleost values, except for pyruvate kinase, citrate synthase, and 3-hydroxyacyl-CoA dehydrogenase, for which activities resembled teleost levels. Enzyme values from the fed fish (7 months) were generally the same as the newly captured group, and food deprivation increased phosphoenolpyruvate carboxykinase activities without altering other enzyme levels. These results support the view that hagfish are anoxia tolerant with low metabolic potential and demonstrate that the muscle and liver rely on carbohydrate and lipid reserves during fasting.

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