Gillian M. Lawrence

The compartmentation of glycolytic and gluconeogenic enzymes in rat kidney and liver and its significance to renal and hepatic metabolism

SummaryAn indirect immunoperoxidase procedure has been used to demonstrate sites of glycolysis and gluconeogenesis in normal rat kidney and liver. In kidney, the gluconeogenic enzyme fructose 1,6-biphosphatase was restricted to the proximal tubular epithelium, while the glycolytic enzyme hexokinase predominated in more distal segments. Intense staining for the biphosphatase in proximal convoluted tubular brush borders suggests that reabsorbed substrates may be used directly at this site in renal gluconeogenesis. In view of the high phosphofructokinase and pyruvate kinase activities present in collecting ducts, their relatively low hexokinase activities and their relatively pale immunostaining for hexokinase indicate that glycolytic substrates which feed into the pathway subsequent to the initial phosphorylation step, rather than glucose, may be the major energy source for the rat renal papilla.Immunostaining in the liver was consistent with the metabolic zonation of liver parenchyma, in that glucokinase occurred mainly in perivenous regions and fructose 1,6-bisphosphatase in periportal areas. The presence of such metabolic zonation is difficult to reconcile with the widely held view that the majority of hepatic glucogen is derived directly from glucose. A model for hepatic glycogen synthesis is proposed which links the concept of parenchymal zonal heterogeneity with recent biochemical evidence concerning the ‘glucose paradox’ and with microscopical studies on the dynamics of glycogen deposition after refeeding.

Histochemical and immunohistochemical localization of hexokinase isoenzymes in normal rat liver

SummaryHistochemical and immunohistochemical procedures have been used to examine the localization of three of the four hexokinase isoenzymes present in the liver of fed female Wistar rats. Distinctive distribution patterns were found for hexokinase type I and glucokinase but hexokinase type II was not detectable. Hexokinase type I was identified in sinusoidal cells and in bile duct epithelia, nerves and arteries in the portal triad. Glucokinase, the major isoenzyme, was confined to parenchymal cells where it was present in much higher amounts in perivenous compared with periportal hepatocytes. Staining within these two zones was not homogeneous and each had a mosaic appearance caused by the presence of a few hepatocytes containing little or no glucokinase amongst the majority of darkly stained cells in perivenous areas and a few darkly stained cells amongst the majority of unstained cells in periportal areas. Hence, hepatocytesin situ are a strikingly heterogeneous population of cells. Their metabolic status cannot be controlled simply by the differential supply of oxygen, substrates and hormones to different regions of the liver acini as proposed in the metabolic zonation model. Phenotypic differences may exist between cells within a given metabolic zone which influence their ability to respond to different environmental conditions.

The localization of hexokinase isoenzymes in red and white skeletal muscles of the rat.

SummaryMaximum assayable hexokinase activities vary with the proportion of red, fast-twitch, oxidative-glycolytic and intermediate, slow-twitch, oxidative fibres in different rat skeletal muscles. The major isoenzymic form, type II hexokinase, is present throughout the intermyofibrillar sarcoplasm in all fibres but a proportion of the total activity appears to be weakly associated with mitochondria. Variations in the histochemical staining intensity between fibre types correlate with their mitochondrial content and seem to be due mainly to differences in mitochondrially-associated hexokinase activity. Changes in the strength of this association may be important in controlling increases in glucose metabolism in response to prolonged increased muscular activity while regulation of the equilibrium between free and loosely-bound forms may be an important control feature in all skeletal muscle. Type I hexokinase is a minor isoenzymic component of skeletal muscle and occurs mainly in blood vessels and nerves in the perimysia and endomysia. The majority of this isoenzyme is tightly bound to mitochondria and is not detectable in homogenates prepared in the absence of Triton X-100.

Antigenic cross-reactivities between mammalian hexokinases

Immunoelectrophoresis and gel diffusion techniques have demonstrated that, contrary to previous reports, antigenic cross-reactivity does exist between the three major rat hexokinase isoenzymes. Immune inhibition and immune precipitation assays performed in parallel generally failed to detect this cross-reactivity and possible reasons for this failure are discussed. The inability of the latter assay systems to detect cross-reactivity would seem to account for the discrepancies between the present results and those obtained in earlier studies.

Histochemical evidence of changes in fuel metabolism induced in red, white and intermediate muscle fibres of streptozotocin-treated rats

SummaryThe present study provides histochemical evidence supporting the operation of the ‘glucose—fatty acid cycle‘ in skeletal muscles taken 5 days after the administration of a single injection of streptozotocin. It also indicates that the cycle is more important in fast-oxidative-glycolytic (FOG) and slow-oxidative (SO) fibres than in fast-glycolytic (FG) fibres. Data from muscles taken 14 and 28 days after treatment suggest that lipid catabolism becomes progressively less important with time, and that muscles from longer-term diabetic rats rely on the aerobic and anaerobic breakdown of glucose by FOG and FG fibres to meet their cellular energy requirements. Although SO fibres appeared initially to be the least affected by steptozotocin-induced diabetes, the decline in their metabolic capabilities ultimately seemed to be greater than that in FOG fibres. Transformations in the biochemical characteristics of FOG and SO fibres occurred 14–28 days after streptozotocin treatment, in the absence of changes in actomyosin-ATPase activity. This supports the view that the division of skeletal muscle fibres into three or four distinct types on the basis of myosin- or actomyosin-ATPase activity is an oversimplification of the true situation.

Hexokinase isoenzymes: antigenic cross-reactivities and amino acid compositional relatedness.

Previously undetected antigenic cross-reactivities have been demonstrated between yeast and rat hexokinase isoenzymes using an enzyme-linked immune sorbent assay. The levels of structural homology between the isoenzymes have been assessed in terms of their relative antigenic cross-reactivities and their amino acid compositional relatedness. The three major rat hexokinases appear, despite their differing molecular sizes, to have a close evolutionary background and to share a common but distant ancestry with yeast and wheat-germ forms.

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity.

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gka and Gkb, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent Km values for MgATP2− and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.

The ubiquitous localization of type I hexokinase in rat peripheral nerves, smooth muscle cells and epithelial cells

SummaryAn indirect immunoperoxidase technique has been used to determine the localization of type I hexokinase in a wide variety of Carnoy-fixed, paraffin-embedded rat tissues. The results suggest that the widespread tissue distribution of the isoenzyme is due to its ubiquitous localization in the nervous, smooth muscle and epithelial components of each tissue. The majority of the immunostaining was confined to cells with substantial energy requirements which are probably mainly satisfied through the breakdown of glucose. This observation is consistent with the known predominance of type I hexokinase in the central nervous system and with the regulatory role allotted to it in this tissue.