Rolf F. Kletzien

Regulation of glucose-6-phosphate dehydrogenase by diet and thyroid hormone

The regulation of hepatic glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) RNA by thyroid hormone and high carbohydrate (sucrose) diet was studied. Previous studies from several laboratories have demonstrated that thyroid hormone modulates G6PDH activity. However, the point at which thyroid hormone exerts this regulation has not been adequately addressed. In order to assess the role of thyroid hormone in this regulation, levels of G6PDH mRNA were determined in hypothyroid rats maintained on normal or high carbohydrate diets with or without thyroid hormone (triiodothyronine; T3) supplementation. A dot-blot hybridization procedure with nick-translated cDNA probes was used to directly assess the relative concentrations of G6PDH mRNA. Enzyme activity increased when the animals were treated with T3 and/or placed on a high carbohydrate diet. However, there was no effect of T3 and diet, alone or in combination, on G6PDH mRNA levels in hypothyroid rats. The data suggests that thyroid hormone and high carbohydrate diet are acting at a translational level to increase G6PDH enzyme activity in these animals.

The effect of nutritional status and of glucocorticoid treatment on the protein kinase isozyme pattern of liver parenchymal cells

In this study, we have employed native polyacrylamide gels to resolve protein kinase isozymes in cytosol samples from rat liver parenchymal cells. Five distinct bands of protein kinase activity were detected. Four of these were found to be cyclic AMP-dependent. Cytosols of isolated hepatocytes from fed and fasted rats were compared using the gel system. The resulting protein kinase activity profile revealed no marked differences in isozyme pattern or kinase activity between the two nutritional states. Isolated hepatocytes from fed rats were placed in primary culture and after 24 and 48 h cytosol samples were prepared and assayed for protein kinase activity using the gel system. The isozyme pattern remained intact for cells maintained in either Waymouths or Swims-77 medium for at least 48 h after inoculation into culture, and the isozyme pattern was not altered by glucocorticoid treatment.

Ethanol-glucocorticoid regulation of hepatic glucose-6-phosphate dehydrogenase

The effect of ethanol, alone and in combination with glucocorticoid and insulin, on glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of rat hepatocytes maintained in a chemically defined medium. Maintenance of hepatocytes from fasted animals in a culture medium devoid of hormones and ethanol resulted in a 2.5-fold increase in G6PDH activity in 48 hr. Parallel cultures treated with glucocorticoid and insulin or glucocorticoid, insulin and ethanol stimulated enzyme activity 6- and 9-fold, respectively in 48 hr. Treatment with ethanol for 48 hr potentiated basal and glucocorticoid plus insulin-induced enzyme activity 1.4-fold. The activity of G6PDH mRNA, estimated by cell-free translation of hepatic mRNA in a mRNA-dependent reticulocyte lysate and by RNA dot-blot hybridization, was compared with enzyme activity and relative rate of G6PDH synthesis. The increases in enzyme activity observed in response to glucocorticoid and insulin or ethanol, alone or in combination with glucocorticoid and insulin, were paralleled by comparable increases in the rate of synthesis and mRNA levels of G6PDH. The results of this study show that the glucocorticoids, insulin and ethanol interact to stimulate the synthesis of G6PDH primarily by increasing the concentration of G6PDH mRNA.

Histochemical Analysis of Hepatocarcinogenesis

The hybrid discipline of histochemistry, a borderline field between histology and analytical chemistry or biochemistry is concerned with the identification, localization and quantification of specific substances, reactive groups and sites of enzymatic activities in tissues, cells and cell organelles. Principally metabolic products or enzymes can also be assessed by biochemical analysis nowadays much more efficiently because of the development of new sensitive microchemical techniques. However, the data provided by this approach represent average values and cannot give any information about the true distribution of certain compounds in individual cells and organells. That individual cells differ markedly in their metabolic compartimentation is best illustrated by the kidney tubular system1 and even the liver which looks quite homogeneous uncovers at closer inspection the well known metabolic zonation2. Biochemical analysis becomes really difficult or cannot be performed at all when patholologically altered tissue has to be investigated in which cellular structure as well as metabolic activities may have changed. Because histochemistry offers a wide spectrum of effective methods to surmount such serious problems it turned out to be an indispensable tool for scientists in many fields from botany to histopathology which is frequently applied in toxicology and cancer research.

Characterization of cytochalasin B binding to adult rat liver parenchymal cells in primary culture

The characterization of cytochalasin B binding and the resulting effect on hexose transport in rat liver parenchymal cells in primary culture were studied. The cells were isolated from adult rats by perfusing the liver in situ with collagenase and separating the hepatocytes from the other cell types by differential centrifugation. The cells were established in primary culture on collagen-coated dishes. The binding of [4-3H]cytochalasin B and transport of 3-O-methyl-D-[14C]glucose into cells were investigated in monolayer culture followed by digestion of cells and scintillation counting of radioactivity. The binding of cytochalasin B to cells was rapid and reversible with association and dissociation being essentially complete within 2 min. Analysis of the kinetics of cytochalasin B binding by Scatchard plots revealed that binding was biphasic, with the parenchymal cell being extremely rich in high-affinity binding sites. The high-affinity site, thought to be the glucose-transport carrier, exhibited a KD of 2.86 x 10(-7) M, while the low-affinity site had a KD of 1.13 x 10(-5) M. Sugar transport was monitored by 3-O-methyl-D-glucose uptake and it was found that cytochalasin B (10(-5) M) drastically inhibited transport. However, D-glucose (10(-5) M) did not displace cytochalasin B, and cytochalasin E, which does not inhibit transport, was competitive for cytochalasin B at only the low-affinity site, demonstrating that the cytochalasin B inhibition of sugar transport occurs at the high-affinity site but that the inhibition is non-competitive in nature. Therefore, the liver parenchymal cells may represent an unusually rich source of glucose-transport system which may be useful in the isolation of this important membrane carrier.