Hans-Dieter Söling

The redox state of NAD+/NADH systems in guinea pig liver during increased fatty acid oxidation

Abstract As fatty acids fail to stimulate gluconeogenesis in guinea pig livers6,7, changes in the redox state of the substrate pairs lactate/pyruvate, 3-hydroxybutyrate/acetoacetate, glutamate/(α-oxoglutarate)(NH4+) and α-glycerophosphate/dihydroxy-acetone phosphate as well as changes in the ATP/ADP ratio were studied in guinea pig livers during increased oxidation of fatty acids in vivo and during liver perfusion. the following results were obtained: 1. 1. Under normal conditions the redox state of the lactate/pyruvate system is more negative, that of the 3-hydroxybutyrate/acetoacetate system more positive in guinea pig liver (in vivo and during isolated perfusion) than in rat liver. The redox state of the α-glycerophosphate/dihydroxyacetone phosphate system is about the same as in rat liver. 2. 2. During increased oxidation of fatty acids, the redox state of the lactate/ pyruvate system in isolated perfused guinea pig livers becomes more negative, whereas the redox state of the 3-hydroxybutyrate/acetoacetate system shows a slight increase only during prolonged fatty acid oxidation. In vivo, fat feeding for 3 days leads to an increase in the lactate/pyruvate ration, to a dramatic increase in the α-glycerophosphate/dihydroxyacetone phosphate ration, and to a doubling of the 3-hydroxybutyrate/acetoacetate ratio. 3. 3. The ATP/ADP ratio in the guinea pig liver is not altered by an increased oxidation of fatty acids. 4. 4. Data are presented which make it unlikely that the activity of 3-hydroxybutyrate dihydrogenase (EC 1.1.1.30) is insufficient to establish the 3-hydroxybutyrate/acetoacetate equilibrium under the experimental condition used. 5. 5. Under normal conditions the redox state of the 3-hydroxybutyrate dehydrogenase system and that of the glutamate dehydrogenase (EC 1.4.1.3) system are in equilibrium in rat and guinea pig liver. Under certain experimental conditions a disequilibrium is observed as well in guinea pig as in rat liver. It is suggested that this results from different compartmentation of some fo the substrates of these redox systems.

Purkinje cells of rat and chicken cerebellum contain calreticulin (CaBP3)

The presence of the calcium‐binding protein calreticulin (CaBP3) was assessed in rat and chicken cerebellum by immunoblotting, and its localization in Purkinje cells was established by immunocytochemistry and in situ hybridization.

Perifusion system for isolated cells

Abstract A system is described for perifusion of isolated hepatic cells which has a small interior space, rapidly responds to substrate changes, and requires no mechanical agitation of the cells. A 22 × 2-mm circular chamber is used with membranes (25 mm diameter, 8 μm pore size) at top and bottom. The solution enters the chamber at the bottom, perifuses the cells, and exits at the top. Gravity prevents cell clogging on the top membrane. Cells equivalent to 110–150 mg wet wt are perifused in a 0.7-ml chamber space. ATP levels, glucose and urea production, glucagon effects, and other measurements indicate excellent cell viability. O2 utilization is directly monitored. Examples of α-aminoisobutyrate and alanine uptake, and lactate and alanine metabolism in hepatocytes from 48-h fasted rats are given.

Ketogenic action of fructose in the isolated perfused rat and guinea pig liver.

During studies with fructose as a precursor for gluconeogenesis in isolated perfused livers from rats and guinea pigs a significant rate of ketogenesis was observed which could not be detected with other gluconeogenic precursors (glycerol, pyruvate, lactate) or with glucose. The data presented in this paper show that fructose enhances the formation of acetyl-S-CoA from pyruvate by stimulating pyruvate oxidation. The stimulation of pyruvate oxidation results from an increased activity of pyruvate dehydrogenase due to the fructose induced fall in the concentrations of adenine nucleotides.

Potenzierung der Wirkung von exogenem Insulin durch N-(4-Methylbenzolsulfonyl)-N'-butylcarbamid und N1,-n-Butylbiguanid beim eviscerierten Tier

Zusammenfassung1. Sulfonylharnstoffe (Tolbutamid und Chlorpropamid) senken an der eviscerierten, nephrektomierten Ratte den Blutzucker nicht und erhöhen nicht den Glucoseverbrauch.2. Tolbutamid (D 860) erhöht die Insulinempfindlichkeit der eviscerierten Ratte deutlich, hat also einen in die Peripherie zu lokalisierenden insulinpotenzierenden Effekt. Dieser Befund kann nicht erklärt werden. Die klinische Erfahrung spricht dagegen, daß er im Wirkungsmechanismus der Sulfonylharnstoffe eine wesentliche Rolle spielt.3. N1,-n-butylbiguanid (W37) senkt den Blutzucker der eviscerierten Ratte mit und ohne Nebennieren nicht und erhöht nicht ihren Glucosebedarf.4. W37 erhöht die Insulinempfindlichkeit der eviscerierten Ratte im gleichen Ausmaß wie D 860. Im Unterschied zum D 860 führt W37 zu einer starken Muskelglykogenolyse. Ob die Potenzierung der Insulinwirkung durch Biguanide eine wesentliche Bedeutung für deren Wirkungsmechanismus hat, ist fraglich.

Metabolite‐controlled phosphorylation of hepatic phosphofructokinase proceeds by cAMP‐dependent protein kinase

In hepatocytes 32P‐incorporation into rat liver phosphofructokinase is stimulated by glucose as well as by glucagon, the effects of both stimuli being prevented by L‐alanine [Eur. J. Biochem. (1982) 122, 175]. The phosphopeptides of the enzyme derived from limited proteolysis by subtilisin and from exhaustive tryptic digestion were analyzed either by one‐dimensional mapping on sodium dodecyl sulphate‐polyacrylamide slab gels and by fingerprint mapping, respectively. It is shown that in vivo stimulation of 32P‐incorporation by glucose or by glucose plus glucagon results in identical phosphopeptide maps, and that these maps were identical with those obtained from phosphofructokinase phosphorylated in vitro with catalytic subunit of cAMP‐dependent protein kinase. It is concluded that in the intact liver cell phosphofructokinase is phosphorylated by cAMP‐dependent protein kinase but that the state of phosphorylation is modified by metabolite control.

Regulation of gluconeogenesis in rat and guinea pig liver.

Summary The stimulation of gluconeogenesis from L-lactate by isolated perfused livers of fed or starved rats by fatty acids is well established. Similar experiments with isolated perfused guinea pig livers showed a significantly higher rate of net formation of glucose from 10 or 20 mM L-lactate compared with rat liver, but hexanoate as well as albumin-bound oleate inhibited the net formation of glucose and the uptake of L-lactate in guinea pig liver under conditions where both parameters were stimulated in rat liver. Glucagon as well as 3′, 5′-AMP or its dibutyryl-derivative failed to stimulate gluconeogenesis from L-lactate in guinea pig liver. Ketogenesis by rat liver from oleate was considerable, that from hexanoate moderately higher compared with guinea pig liver, but differences with respect to glucose formation remained even under conditions of similar rates of ketogenesis. Changes of the levels of acetyl-S-CoA and free CoA-SH during stimulated fatty acid oxidation were similar in both species. An analysis of activities of enzymes involved in gluconeogenesis and glycolysis in the fed and the 48-hour starved state revealed in guinea pig liver a significantly lower glucokinase + hexokinase/glucose-6-phosphatase ratio and a significantly higher phosphofructokinase/FDPase ratio. The pyruvate kinase/pyruvate carboxylase ratio was 4.00 in the fed (rat: 9.50), 4.75 (rat: 3.22) in the 48-hour starved state. The pyruvate kinase/phosphoenolpyruvate carboxykinase ratio was 0.65 in the fed (rat: 11.90), 0.82 (rat: 4.55) in the 48-hour starved state. Partially purified pyruvate carboxylase from guinea pig and rat livers behaved similarly with respect to apparent KM′s for pyruvate and ATP and to the apparent Ka for acetyl-S-CoA at different pH values, but exhibited different pH optima with Mn++ (rat: 8.05, guinea pig: 7.25) or Mg++ (rat: 8.50, guinea pig: 7.95). It seems unlikely that pyruvate carboxylation is a site of regulation of gluconeogenesis in guinea pig liver. Inhibition of gluconeogenesis from L-lactate by 5-methoxy-indol-2-carbonic acid and the release of this inhibition by fatty acids occurred similarly in rat and guinea pig livers. Quinolinic acid, on the other hand, exhibited a much stronger inhibitory effect in rat liver than in guinea pig liver, while it was without any effect at all in the isolated perfused pigeon liver. Studies with 14C-quinolinic acid showed that this agent does not enter the mitochondrial compartment at a measurable rate. The degree of inhibition of gluconeogenesis seems to be inversely related to the proportion of phosphoenolpyruvate carboxykinase located in the mitochondria.

Über die Autoregulation des Glomerulusfiltrates bei intratubulärem Druckanstieg am Hund

Zusammenfassung1. Bei Hunden kommt es unter der Bedingung einer normalen Diurese nach Ureterabklemmung zur Entwicklung des sog. „hohen“ Ureterdruckes, dessen Plateau Ausdruck eines Gleichgewichtes von Glomerulusfiltration und tubulärer Rückresorption ist, weil das mit Inulin indirekt bestimmte Glomerulusfiltrat gegenüber dem Ausgangswert entweder unverändert oder nur reduziert gefunden wurde.2. Unter osmotischer Diurese kommt es nach Ureterabklemmung zur Ausbildung des „maximalen“ Ureterdruckes, bei dem nicht mehr filtriert werden kann.3. Der intratubuläre Druckanstieg führt zu einer Zunahme des intraglomerulären Capillardruckes, so daß während des „hohen“ Ureterdruckes der effektive Filtrationsdruck unverändert gefunden werden kann. Der „maximale“ Ureterdruck übersteigt den „hohen“ um den bisherigen Betrag des effektiven Filtrationsdruckes.4. Die Zunahme des intraglomerulären Capillardruckes (Autoregulation des Glomerulusfiltrates) ist die Folge einer venösen Abflußbehinderung durch druckbedingte Erweiterung der Tubuluslumina und die Folge einer Erweiterung des Vas afferens.5. Das Ausmaß der Erweiterung des Vas afferens bestimmt das Verhalten der Nierendurchblutung bei intrarenalem Druckanstieg durch Harnabflußbehinderung. Die präglomeruläre Widerstandsabnahme kann trotz gesteigerter intrarenaler Drucke die postglomeruläre Widerstandszunahme kompensieren — oder sogar überwiegen, so daß dann die Nierendurchblutung zunimmt.6. Es wird dieDilatation des Vas afferens beiintrarenalem (intratubulärem) Druckanstieg gegenübergestellt derConstriction dieses Gefäßes beiintraarteriellem Druckanstieg (Autoregulation der Nierendurchblutung).

Die Beziehungen zwischen Durchblutung und Sauerstoffverbrauch bzw. arterio-venöser Sauerstoffdifferenz in der Hundeniere

Zusammenfassung1. Der O2-Verbrauch der Niere bei Hunden von 9–18 kg beträgt bei genügender Zirkulation 0,03–0,09 ml O2/g × min. Die O2-AV-Diff. ist dann 0,7–3,9 Vol-% O2.2. Erniedrigung der Prozent-O2-Sättigung sowie Manipulationen an der Niere erhöhen den O2-Verbrauch.3. Die O2-AV-Diff. in der Niere ist im Gleichgewichtszustand durchblutungsabhängig. Nur als Sonderfall wird eine konstante O2-AV-Diff. bei Stromstärkeänderungen beobachtet. Der O2-Verbrauch der Niere ist also wie die Wärmebildung bei genügender Zirkulation unabhängig von der Durchblutung.1. Der O2-Verbrauch der Niere bei Hunden von 9–18 kg betragt bei genugender Zirkulation 0,03–0,09 ml O2/g × min. Die O2-AV-Diff. ist dann 0,7–3,9 Vol-% O2.

Differentially high ureteral pressure in relation to the form of diuresis in dogs

ZusammenfassungBei Vorliegen einer „normalen“ Diurese wird nach Ureterabklemmung der sog. „hohe“ Ureterdruck, unter osmotischer Diurese der „maximale“ erreicht. Die Differenz von Blutdruck und „maximalem“ Ureterdruck war im Mittel der Versuche um 20 mm Hg kleiner als diejenige des „hohen“. Die Ursache dafür wird kurz diskutiert.Bei Vorliegen einer „normalen“ Diurese wird nach Ureterabklemmung der sog. „hohe“ Ureterdruck, unter osmotischer Diurese der „maximale“ erreicht. Die Differenz von Blutdruck und „maximalem“ Ureterdruck war im Mittel der Versuche um 20 mm Hg kleiner als diejenige des „hohen“. Die Ursache dafur wird kurz diskutiert.