C-peptide attenuates protein tyrosine phosphatase activity and enhances glycogen synthesis in L6 myoblasts

Recent studies suggest that C-peptide might play a role in a broad range of biological activities. We have provided evidence that C-peptide stimulates glycogen synthesis in insulin-responsive rat skeletal muscle cells in a dose-related manner. To explore the mechanism by which C-peptide exerts this insulinomimetic effect, here we report the effect of C-peptide on protein tyrosine phosphatase (PTP) activity and phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1). C-peptide inhibited PTP activity in a dose-dependent manner. A reverse bell-shaped dose-response curve was shown with the maximum inhibition of PTP activity at a concentration of 3 nM of C-peptide, which is the same concentration achieving the maximum stimulatory effect on glycogen synthesis. In association with the PTP inhibition by C-peptide, autophosphorylation of the insulin receptor and activation of IRS-1 were enhanced. These results suggest that C-peptide signal transduction may crosstalk with the insulin signaling pathway at the level of the insulin receptor.     

Single motoneuron succinate dehydrogenase activity

We have developed a quantitative histochemical assay for measurement of succinate dehydrogenase (SDH) activity in single motoneurons. A computer image processing system was used to quantify the histochemical enzyme reaction product and to follow the time course of the reaction. The optimal concentration for each of the ingredients of the incubation medium for the SDH reaction was determined and the importance of using histochemical "blanks" in the determination of enzymatic activity was demonstrated. The enzymatic activity was linear with respect to reaction time and tissue thickness. The procedure described meets the criteria generally considered essential for establishment of a quantitative histochemical assay. The assay was then used to examine the SDH activity of cat and rat motoneurons. It was found that motoneurons with a small soma size had a wide range of SDH activity, whereas those with a large soma size were restricted to low SDH activity.     

Maxi chloride channels in L6 myoblasts.

The existence of a large conductance voltage sensitive chloride channel is documented in undifferentiated cells (myoblasts) of the L6 rat muscle cell line. At this stage of development the resting membrane conductance is dominated by potassium ions only (Kidokoro 1975). The conductance of the channel in symmetrical 120 mmol/l choline chloride is 331 +/- 4 pS. The probability of the channel being in the open state decreases with the increasing imposed voltage. Due to rapid inactivation at high membrane potential deviations (both negative and positive) from the equilibrium potential the channel can be resolved clearly by pulse technique protocols only. The incidence of the channel in successful patch trials was higher than usually reported. The channel was present after differentiation of the myoblasts into the myotubes. It showed at least one definite substate and pronounced flickerings between the substate and the main open state. The channel was observed in myoblast attached patches as well. It is supposed to belong to the category of maxi chloride channels, and to play probably a role in regulatory volume readjustment or in cell communication during myogenesis, respectively.     

Lysosomal triacylglycerol lipase activity in L6 myoblasts and its changes on differentiation.

L6 myoblasts, before fusion, accumulate large stores of neutral lipid when cultured in medium supplemented with fatty acid. Upon fusion to terminally differentiated myotubes, a noticeable decrease in these neutral-lipid stores was observed. Triacylglycerol lipase activity was examined in L6 myoblasts at various stages of cell differentiation to assess a possible role for this enzyme in the above phenomenon. In this first study to demonstrate lipolytic activity in cultured muscle cells, the activity was found to be totally dependent on the presence of a detergent, either Cutscum or Triton X-100, during homogenization. The inhibition by many thiol-specific reagents [N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5'-dithiobis-(2-nitrobenzoic acid)] suggest that a thiol group is at or near the active site. The observed acidic pH optimum (5.5-6.0), the acute inhibition by chlorpromazine (a lysosomal lipase inhibitor) and the distribution of lipolytic activity upon cell fractionation (which co-sediments with acid phosphatase, a lysosomal marker enzyme) suggest that the lipase may be of lysosomal origin. Under the optimal conditions described, the triacylglycerol lipase activity of L6 myoblasts was determined to be 2.9 +/- 0.4 nmol of oleic acid released/min per mg of DNA. This activity increased 3-fold, to 9.0 +/- 1.6 nmol/min per mg, in the myotube phase. This increase in lipolytic activity may be responsible for the observed decrease in neutral-lipid stores of differentiating myoblasts.     

A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP⁺ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.     

Electron microscopic-cytochemical heterogeneity of succinate dehydrogenase activity in rat cardiomyocytes

The ultrastructural localization of succinate dehydrogenase in white rat heart myocytes is studied and heterogeneity of reaction products in separate mitochondria and their groups is described. The enzyme activity is cardiomyocyte electron density dependent. This dependence, in all probability, is the result of different structural and functional states of cells and their organelles, that is revealed by electron microscopy as different electron density of these. It is found that middle electron density cells have the maximum enzyme activity. The mechanisms of enzyme activity dependence of cell electron density are discussed.     

Glucose-6-phosphate dehydrogenase and succinate dehydrogenase in metastatic cells of Lewis lung carcinoma

By the experimental model of the Lewis lung carcinoma we studied the activity of glucose-6-phosphate-dehydrogenesis (G6PDH) and succinic-dehydrogenesis (SDH) in the carcinoma itself, the lung metastases, the peripheral blood. A set of carcinoma, lung sections, peripheral blood smears were studied by histochemical techniques day by day since the 3rd day to the 15th one after the carcinoma grafting. The reported results allow us to conclude that G6PDH looks like a determinant of metastatic cell survival and growth and can be regarded as a marker of metastatic cells in the present model.     

Morphological characterization of actively fusing L6 myoblasts

We have characterized morphologically the surface of L6 myoblasts at the time of active cell fusion using transmission electron microscopy. Two subclones of the L6 line were used in these studies: the L6Cl55 line that fuses to form multinucleated syncytia and the NF44 non-fusing variant. Ultrastructural analysis revealed an electron-opaque material at localized points of cell-cell apposition in actively fusing L6Cl55 cells. This material may be transported by and secreted from smooth-surfaced cytoplasmic vesicles with an electron-dense core. In contrast to L6Cl55 cells, the electron-dense plaques were seen infrequently in cultures of the NF44 non-fusing variant. This previously unidentified substance may be associated with cell-cell recognition or adhesion, both necessary prerequisites for myoblast membrane fusion. Alternatively, the electron-dense plaques may be directly involved in the fusion event.     

Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity

Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.     

Rhodanese-Mediated sulfur transfer to succinate dehydrogenase.

The interaction of the sulfurtransferase rhodanese (EC 2.8.1.1) with succinate dehydrogenase (EC 1.3.99.1), yeast alcohol dehydrogenase (EC 1.1.1.1) and bovine serum albumin was studied. Succinate dehydrogenase incorporates the sulfane sulfur of [35S]rhodanese and, in the presence of unlabelled rhodanese, also incorporates that of [35S]thiosulfate. Rhodanese releases most of its transferable sulfur and is re-loaded in the presence of thiosulfate. Rhodanese undergoes similar modifications with yeast alcohol dehydrogenase but this latter does not bind 35S in amounts comparable to those incorporated in succinate dehydrogenase: nearly all the 35S released by [35S]rhodanese is with low-molecular-weight compounds. Bovine serum albumin also binds very little sulfur and [35S]rhodanese present in the reaction mixture does not discharge its radioactive sulfur nor does it take up sulfur from thiosulfate. Sulfur release from rhodanese appears to depend on the presence of - SH groups in the acceptor protein. Sulfur incorporated into succinate dehydrogenase was analytically determined as sulfide. A comparison of the optical spectra of succinate dehydrogenase preparations incubated with or without rhodanese indicates that there is an effect of the sulfurtransferase on the iron-sulfur absorption of the flavorprotein. The interaction of rhodanese with succinate dehydrogenase greatly decreases the catalytic activity of rhodanese with respect to thiocyanate formation. This is attributed to modifications in rhodanese associated with the reduction of sulfane sulfur to sulfide. Thiosulfate in part protects from this deactivation. The reconstitutive capacity of succinate dehydrogenase increased in parallel with sulfur incorporated in that enzyme following its interaction with rhodanese.     

Tight binding of oxaloacetate to succinate dehydrogenase

[¹⁴C]Oxaloacetate forms a stable complex with succinate dehydrogenase which withstands repeated Sephadex filtration. Oxidized glutathione, 2-thenoyltrifluoroacetone, KCN and ageing at +4° at neutral pH do not prevent the enzyme to bind oxaloacetate. The binding is prevented by succinate or malonate but the complex, once formed, can not be split by these compounds, although the enzyme activity can be restored; the reconstitutive property of succinate dehydrogenase is, however, irreversibly lost. Bound oxaloacetate does not exchange with added oxaloacetate, but can be released by perchloric acid. Sonic particles of beef heart mitochondria can also bind oxaloacetate. However, this complex can be split by succinate or malonate.     

The radial distribution of succinate dehydrogenase activity in porcine muscle fibres

Summary A microscope photometer with a computer-controlled scanning stage was used to map the distribution of succinate dehydrogenase (SDH) activity in transverse sections of skeletal muscle fibres of pigs from 9 to 29 weeks of age. Absorbance due to SDH activity was measured in successive concentric zones that converged on the central axis of the muscle fibre. In all fibres, there was less SDH activity in the axis of the fibre than in the periphery of the fibre. In fibres with strong adenosine triphosphatase (ATPase) activity and weak overall SDH activity, the radial gradient of SDH activity remained constant as fibres grew in cross-sectional area. However, in fibres with weak ATPase and strong SDH and in fibres with strong ATPase and strong SDH, the radial gradient of SDH activity increased as fibres grew larger. Changes in radial gradients were due to both decreased SDH activity in the axis and to increased SDH activity in the periphery. In all three fibre types, the overall SDH activity per fibre increased with age.     

Studies in succinate dehydrogenase histochemistry

Summary The activity of succinate tetrazolium reductase was investigated in liver and kidney from the rat and mouse. The results obtained were related to the cellular level of succinate dehydrogenase (SDH) as well as to the level of CoQ.It was concluded that the low activity in centrolobular areas of the liver lobules compared with the perilobular areas, exclusively is due to a naturally deprivation of CoQ.The level of SDH as well as of CoQ was very high in renal cortical tubules rich in mitochondria (proximal and distal convoluted tubules, the ascending thick limb of Henle). This was indicated by the facts that the initial reaction rate was high and no enhancement was obtained by the addition of CoQ₁₀.In all experiments the activity of fresh frozen sections were compared with the activity of sections from briefly prefixed tissue. The influence of different fixatives, variation in Nitro BT concentration, cryoprotection (dimethyl sulfoxide, DMSO) and osmolar protection (sucrose) was investigated and discussed. Further, the substrate-carrying effect of DMSO was investigated and discussed.Brief (5 min) fixation at 0–4° C—especially with 1% buffered (pH=7.2) methanol-free formaldehyde (from paraformaldehyde) gave excellent preservation of morphology and caused no inhibition of SDH activity. Furthermore, the fixation caused an enhancement of Nitro BT penetration into the tissue and an enhancement of formazan substantivity.The incubation time needed for the appearance of both the red and blue formazan was recorded in order to follow the initial reaction rate. This procedure proved to be a sensitive indicator, when the influence of components added (CoQ₁₀, DMSO, sucrose etc.) was studied.