A novel method for continuous online glucose monitoring in humans: the comparative microdialysis technique.

The aim of this study was to prove the feasibility of continuous subcutaneous glucose monitoring in humans using the comparative microdialysis technique (CMT). The performance of the CMT was determined by comparing tissue glucose values with venous or capillary blood glucose values in healthy volunteers and type 1 diabetic subjects. The CMT is a microdialysis-based system for continuous online glucose monitoring in humans. This technique does not require calibration by the patient. Physiological saline with glucose (5.5 mM) is pumped in a stop-flow mode through a microdialysis probe inserted into the abdominal s.c. tissue. Tissue glucose concentration is calculated by comparing the dialysate and perfusate glucose concentrations. The time delay due to the measurement process is 9 min. We tested the CMT on six healthy volunteers and six type 1 diabetic patients for 24 h in our clinical setting. Comparisons were made to HemoCue analyzer (Angelholm, Sweden) capillary blood glucose measurements (healthy volunteers) and to venous blood glucose concentration determined with a Hitachi analyzer (diabetic patients). The mean absolute relative error of the CMT glucose values from the blood glucose values was 17.8+/-15.5% (n = 167) for the healthy volunteers and 11.0+/-10.8% (n = 425) for the diabetic patients. The mean difference was 0.42+/-1.06 mM (healthy volunteers) and -0.17+/-1.22 mM (diabetic patients). Error grid analysis for the values obtained in diabetic patients demonstrated that 99% of CMT glucose values were within clinically acceptable regions (regions A and B of the Clarke Error Grid). The study results show that the CMT is an accurate technique for continuous online glucose monitoring.

Effect of starvation or treatment with corticosterone on the amount of easily releasable myofilaments in rat skeletal muscles.

Treatment of isolated myofibrils with an ATP-containing relaxing solution results in the dissociation of a preformed quantity of myofilaments called 'easily releasable myofilaments'. Van der Westhuyzen, Matsumoto & Etlinger [(1981) J. Biol. Chem. 256, 11791-11797] presented experimental evidence that these myofilaments represent intermediate products in the turnover of myofibrillar proteins. To investigate further this question, we measured the size of the fraction of easily releasable myofilaments in three different species of skeletal muscles from rats subjected to well-defined catabolic conditions, namely starvation or chronic glucocorticoid administration. The results were as follows: (1) The amount of easily releasable myofilaments was transiently increased about 2-3-fold during both experiments, and thus paralleled the known alterations in the rate of overall muscle protein breakdown rather than in those of synthesis. (2) These changes were observed in muscles containing predominantly fast-twitch fibres, but not in slow-twitch soleus muscle, a muscle that is known to be more resistant to catabolic conditions. (3) The starvation-induced increase of the size of the fraction of easily releasable myofilaments could be significantly reduced by treatment of the starving animals with the proteinase inhibitor E-64. These results are compatible with the idea that easily releasable myofilaments are intermediates in the degradative pathway of myofibrillar proteins and that a proteolytic step may be involved in the conversion of myofilaments into easily releasable myofilaments.

Loss of fast-twitch isomyosins in skeletal muscles of the diabetic rat.

By means of pyrophosphate electrophoresis the myosin isoenzyme pattern of two fast-twitch skeletal muscles (extensor digitorum longus, gastrocnemius) and one slow-twitch muscle (soleus) was investigated in control rats and was compared with that of rats 4 weeks after induction of diabetes mellitus by streptozotocin injection. In the fast-twitch muscles the isomyosin pattern consisting of FM1 (fast isomyosin 1), FM2 and FM3 was strongly affected by diabetes, resulting in an extensive loss of FM1 and a substantial decrease of FM2. These changes were also apparent when the light chains of the fast isomyosins were analysed by two-dimensional electrophoresis: LC3f (myosin light chain 3f) largely disappeared and LC2f was significantly diminished. In contrast, the isomyosin pattern in soleus muscle, consisting of SM1 (slow isomyosin 1) and SM2, was not affected by the diabetic state, and two-dimensional electrophoresis revealed a normal light-chain pattern of LC1sa, LC1sb and LC2s. These results indicate that the isomyosins of slow-twitch oxidative myofibres are more resistant to the hormonal and metabolic disorders during diabetes mellitus than are the isomyosins of fast-twitch fibres.

Activity of a gelsolin-like actin modulator in rat skeletal muscle under protein catabolic conditions.

A gelsolin-like actin-modulating protein was isolated from rat skeletal muscle and characterized with respect to its interaction with actin. The protein, with a molecular mass of approx. 85 kDa, forms a stoichiometric complex with two actin molecules and is activated by micromolar concentrations of Ca2+. It effectively severs actin filaments and promotes nucleation of actin polymerization. The activity of this protein is detectable already in crude extracts by its capability to reduce the steady state viscosity of actin. Actin-modulating activities were determined in muscle extracts of rats kept under protein catabolic conditions, i.e. as generated by corticosterone treatment and starvation. In both cases we found a marked increase of modulator activity. The possibility is discussed that the increased activity of actin modulator indicates a fragmentation of actin filaments prior to the proteolytic degradation of actin.

Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors.

Three different serine proteinase inhibitors were isolated from rat serum and purified to apparent homogeneity. One of the inhibitors appears to be homologous to alpha 1-proteinase inhibitor isolated from man and other species, but the other two, designated rat proteinase inhibitor I and rat proteinase inhibitor II, seem to have no human counterpart. alpha 1-Proteinase inhibitor (Mr 55000) inhibits trypsin, chymotrypsin and elastase, the three serine proteinases tested. Rat proteinase inhibitor I (Mr 66000) is active towards trypsin and chymotrypsin, but is inactive towards elastase. Rat proteinase inhibitor II (Mr 65000) is an effective inhibitor of trypsin only. Their contributions to the trypsin-inhibitory capacity of rat serum are about 68, 14 and 18% for alpha 1-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor II respectively.

An economic volt-hour integrator, compatible with commercial and homemade electrophoresis power supplies.

In isoelectric focusing systems--a technique widely used for separation and characterization of proteins--the value of the volt-hour integral required to achieve steady-state focusing conditions differs markedly for different proteins. A convenient method to measure the correct volt-hour integral for a given protein, thereby assuring intra- and interlaboratory reproducibility of experiments, can be performed with a volt-hour integrator. As commercially available integrators are compatible only with the power supply marketed by the same manufacturer, a volt-hour integrator was designed which can be used in conjunction with any commercial or homemade power supply. This simple, low-cost integrator is expected to be of interest to those researchers who have so far refrained from introducing volt-hour integration, due to incompatibility of commercial volt-hour integrators with their isoelectric focusing system.

Studies on the alkaline proteinase system in rat skeletal muscle.

The alkaline proteolytic activity, as measured with azocasein as substrate, is significantly increased in the musculus extensor digitorum longus 8 days after induction of diabetes mellitus, whereas in soleus muscle, a slightly lower activity is measured when compared to control rats. Ethyleneglycol extraction of the activity from muscles of normal rats and subsequent fractionation by gel filtration revealed three distinct proteinase activities, corresponding to mol. wts. of 120000, 70000 and 18000. Induction of diabetes mellitus in rats is followed by a loss of the 120000 mol.wt. component. Treatment of the diabetic rats with compound 48/80 results in a complete loss of all three proteinase peaks. The activity of a proteinase inhibitor with an apparent mol. wt. of 60000 was unaffected by these manipulations.

Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle.

A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.

Activation of the multicatalytic proteinase from rat skeletal muscle by fatty acids or sodium dodecyl sulphate.

A multicatalytic proteinase from rat skeletal muscle contains active site(s) catalysing the degradation of benzoyl-Val-Gly-Arg 4-methyl-7-coumarylamide, succinyl-Ala-Ala-Phe 4-methylcoumarylamide and [14C]methylcasein as well as benzyloxy-carbonyl-Leu-Leu-Glu 2-naphthylamide. These activities are 7-14-fold activated by 1 mM-sodium dodecyl sulphate. The activation leads to a higher susceptibility to the proteinase inhibitor chymostatin and to a lower ability to be inhibited and precipitated by antibodies raised against the non-activated enzyme. Since no changes in Mr or subunit composition were observed in the SDS-activated form, some conformational changes seem to occur during the activation step. More pronounced activation was observed in the presence of physiological concentrations of fatty acids; oleic acid at 100 microM concentrations stimulated the proteinase about 50-fold. In contrast with the non-activated proteinase, the activated enzyme considerably degrades muscle cytoplasmic proteins in vitro. Thus it is not unlikely that, in vivo, potential activators such as fatty acids can induce the multicatalytic proteinase to participate in muscle protein breakdown.