Acute gliclazide administration enhances glucose and ketone body utilization in the perfused hind limb of normal and streptozotocin-diabetic rats

Sulfonylureas are the most commonly used oral hypoglycemic agents. Their hypoglycemic actions are produced not only by stimulating insulin secretion but also by extrapancreatic mechanisms. Some groups have already demonstrated the extrapancreatic actions of sulfonylureas on carbohydrate metabolism in the liver, fat and muscle. In this study, we showed in an in situ perfused hind limb preparation of STZ-diabetic rats that gliclazide has an acute effect on ketone body and glucose utilization.     

Enzymatic semisynthesis of porcine despentapeptide (B26-30) insulin using unprotected desoctapeptide (B23-30) insulin as a substrate. Model studies

Unprotected porcine desoctapeptide(B23-30) insulin (DOPI) and the synthetic Gly-Phe-Phe were used as substrates for the trypsin-catalyzed synthesis of despentapeptide(B26-30) insulin (DPPI). The DPPI synthesis was accompanied by a moderate oligomerization and by the formation of a side produce which was identified as a DOPI derivative having an extra peptide bond between the Gly(A1) and Arg(B22) and which was named des(23-63) proinsulin (1). Despite side reactions, the conditions were found where the overall DPPI yields were comparable to those obtained via di-Boc DOPI, and these procedures were faster and simpler since the Boc protection and deprotection steps were omitted. The reaction progress was directly monitored by HPLC.     

Ontogeny of limb force distribution in squirrel monkeys (Saimiri boliviensis): insights into the mechanical bases of primate hind limb dominance

The distribution of peak vertical forces between the forelimbs and the hind limbs is one of the key traits distinguishing primate quadrupedal locomotion from that of other mammals. Whereas most mammals generate greater peak vertical forelimb forces, primates generate greater peak vertical hind limb forces. At the ultimate level, hind limb dominance in limb force distribution is typically interpreted as an adaptation to facilitate fine-branch arboreality. However, the proximate biomechanical bases for primate limb force distribution remain controversial. Three models have been previously proposed. The Center of Mass (COM) Position model attributes primates’ unique mode of limb loading to differences in the position of the whole-body COM relative to the hands and feet. The Active Weight Shift model asserts that primates actively redistribute body weight to their hind limbs by pitching the trunk up via the activation of hind limb retractor muscles. Finally, the Limb Compliance model argues that primates selectively mitigate forelimb forces by maintaining a compliant forelimb and a flat shoulder trajectory. Here, a detailed dataset of ontogenetic changes in morphology and locomotor mechanics in Bolivian squirrel monkeys (Saimiri boliviensis) was employed as a model system to evaluate each of these proposed models in turn. Over the first 10 months of life, squirrel monkeys transitioned from forelimb dominant infants to hind limb dominant juveniles, a change that was precipitated by decreases in peak vertical forelimb forces and increases in peak vertical hind limb forces. Results provided some support for all three of the models, although the COM Position and Active Weight Shift models were most strongly supported by the data. Overall, this study suggests that primates may use a variety of biomechanical strategies to achieve hind limb dominance in limb force distribution.     

Synthesis of an open-chain asymmmetrical cystine peptide corresponding to the sequence A18-21--B19-26 of bovine insulin by solid phase fragment condensation

An insulin fragment containing residues A 18-21 and B 19-26 linked by the disulfide bond between residues A 20 and B 19 was synthesized. The sequence B 21-26 was assembled on a solid support by the Merrifield technique. The protected fragments A 18-21 and B 19-20 were prepared by conventional methods. After forming the disulfide bridge through cleavage of the S-thiocarbonate derivative of A 18-21 by the thiol peptide B 19-20, the resulting assymmetrical cystine peptide A 18-21--B 19-20 was coupled via the carboxyl group of residue B 20 to the free NH 2-terminal amino group of the protected B 21-26 resin. The product was deprotected, cleaved from the resin, and purified to give the homogenous dodecapeptide A 18-21--B 19-26.     

Degradation of oxidized insulin B chain by the multiproteinase complex macropain (proteasome)

The peptides generated from the degradation of the oxidized B chain of bovine insulin by the multiproteinase complex macropain (proteasome) have been analyzed by reverse-phase peptide mapping and identified by N-terminal amino acid sequencing and composition analysis. Six of the 29 peptide bonds in the insulin B chain were found to be rapidly cleaved by macropain. The catalytic center that cleaves the Gln4-His5 bond could be distinguished from the center or centers that cleave the other preferred bonds by its specific susceptibility to inhibition by leupeptin, antipain, chymostatin, and pentamidine, suggesting that macropain utilizes at least two distinct catalytic centers for the degradation of this model polypeptide. The same effectors simultaneously enhance the rate of cleavage at the other susceptible sites in insulin B. The quantitative characteristics of this effect indicate that different catalytic centers of the complex may be functionally coupled, possibly by an allosteric mechanism or possibly by a mechanism in which binding to the catalytic centers is preceded by a rate-limiting binding of the substrate to a site or sites on the enzyme distinct from the catalytic centers. The kinetics of insulin B chain degradation indicate that macropain can catalyze sequential hydrolysis of peptide bonds in a single substrate molecule via a reaction pathway that involves channeling of peptide intermediates between different catalytic centers within the multienzyme complex. This capacity for channeling may confer potential physiological advantages of increasing the efficiency of amino acid recycling and reducing the pool sizes of peptide intermediates that are generated during the degradation of polypeptides in the intracellular milieu.     

B-chain shortening of matrix-bound insulin by pepsin, I:Preparation and properties of bovine des-pentapeptide(B26-30) insulin (suthor's transl)]

Insulin was adsorbed to a strongly acidic ion exchanger and incubated with pepsin. The digestion of the matrix-bound insulin was found to be restricted to the cleavage of the peptide bond between phenylalanine-B25 and tyrosine-B26. Factionation of the reaction products was achieved by gel filtrationon Sephadex G-50 at pH 8 where des-pentapeptide(B26-30)-insulin does not aggregate. Another way to purify this compound was ion-exchange chromatography, which was easy due to the loss of one positive charge on the modified insulin. Crystallization could be achieved in a phenol-containing buffer. Des-pentapeptide(B26-30)-insulin was found to be molecularly uniform by electrophoresis at pH 2.2 and 8.6, thin-layer chromatography, performic acid oxidation, end group analysis and amino acid analysis. The CD-spectrum indicated conformational changes compared to insulin. The biological activity was considerably reduced: fat cell assay 20%, blood sugar depression 30%.     

Effects of insulin and glucose on the production and utilization of glucose in sheep (Ovis aries)

1. The rates of appearance (RA) and metabolic clearance rates (MCR) of glucose were determined during the infusion of insulin plus glucose in sheep. 2. During euglycaemia 50% inhibition of RA of endogenous glucose occurred at insulin concentrations of 60 microU/ml in arterial plasma. 3. During euglycaemia the MCR of glucose doubled with an increment of about 100 microU/ml of insulin. 4. Hypoglycaemia was associated with a reduction in insulin's suppression of RA of endogenous glucose and hyperglycaemia significantly reduced the increase in MCR due to insulin.     

Effects of insulin treatment on ketone body production and carnitine-palmitoyl-transferase (CPT) activity in the isolated perfused liver from streptozotocin diabetic rats

The aim of the present paper was to evaluate the effects of in vivo insulin treatment of streptozotocin (SZ) diabetic rats on the metabolism of the isolated, perfused liver. Perfused livers from SZ-diabetic rats showed a higher ketone body production and a higher mitochondrial carnitine-palmitoyl-transferase (CPT) activity than controls, while triglyceride (TG) output and free-fatty-acid (FFA) uptake were significantly reduced. In vivo insulin treatment normalized both the ketogenic capacity of the liver and CPT activity, while FFA uptake and TG production were still lower than in controls. A significant correlation was found between total ketone body output and CPT activity. We suggest that In vivo insulin treatment of SZ-diabetic rats can modulate the ketogenic capacity of the isolated, perfused liver.     

Effects of diet on catabolism and excretion of (26-14C)cholesterol in rats.

The catabolism and excretion of [26-14C]cholesterol was studied in rats on semisynthetic and commercial diets low in fat or containing 15% butter or corn oil. Rats on the low fat commercial diet oxidized the labeled cholesterol to 14CO2 at more than twice the rate of those on the semisynthetic diet. Fecal excretion of labeled lipid was also somewhat higher with the commercial diet. The added fats had little effect on rate of oxidation of cholesterol but dietary corn oil stimulated fecal excretion of labeled lipid. The rate of loss of labeled cholesterol through oxidation and excretion showed a positive correlation with cholesterol biosynthesis, as measured previously by acetate incorporation into cholesterol in rats on the same kinds of diet. A simple method for efficient trapping and counting of 14CO2 was developed, which facilitated measurement of low levels of 14CO2 in expired air. Estimation of bile acid production from the rate of oxidation of [26-14C]cholesterol to expired 14CO2 and the specific activity of plasma cholesterol gave somewhat higher values than those obtained by other methods. Possible reasons for this difference are discussed.     

Investigation into the mechanism of (-)-epigallocatechin-3-gallate-induced precipitation of insulin

The molecular interactions between EGCG and insulin were investigated to probe the mechanism of EGCG-induced insulin precipitation. The results indicated that 1-5mM EGCG induced insulin into reversible globular precipitates of 185-365 nm. The formation of precipitates was facilitated at high salt concentration and pH values close to insulin's isoelectric point, indicating that hydrophobic interaction was the main driving force. The precipitation was positively related to insulin concentration, but for EGCG, there was a suitable concentration (2 mM at 2 mg/mL of insulin) at which the precipitate content reached maximum. Mass spectroscopy analysis indicated that EGCG formed clusters in the aqueous solution and the clusters correlate with the insulin precipitation. Based on extensive investigation, a physical model was proposed to explain the molecular interactions between EGCG and insulin. Namely, EGCG monomers and clusters first bound to insulin dimers via hydrophobic interaction, leading to the reduction of the thickness of the hydration layer and the partial denaturation of insulin. Then, EGCG clusters acted as bridges to induce the aggregation and precipitation of insulin.     

Hydrolysis of p-nitrophenyl acetate by the peptide chain fragment (336-449) of porcine pancreatic lipase

The incubation of porcine pancreatic lipase (449 amino acids) with chymotrypsin led to the preferential cleavage of the Phe-335-Ala-336 bond [Bousset-Risso et al. (1985) FEBS Lett. 182, 323-326]. Up to now it has not been possible to isolate the fragment (1-335) whereas fragment (336-449) was purified. This fragment does not display any activity towards the specific substrates of lipase, triacylglycerols, either in the aggregate form or monomeric solution, but like lipase it hydrolyzes p-nitrophenyl acetate. The biphasic kinetics of the release of p-nitrophenol by the fragment with different concentrations of p-nitrophenyl acetate ([S] greater than [E]) are very similar to those of lipase and other esterases. The initial burst is equal to 1 mol p-nitrophenol/mol fragment (when [S] = infinity). Ethoxyformic anhydride only reacts with 1 mol histidine out of the 2 mol that the fragment contained. The activity of the fragment towards p-nitrophenyl acetate hydrolysis is inhibited after ethoxyformic anhydride reaction as in the case of lipase. The results presented led to the hypothesis that in the area (336-449) a part of the active-site structure of the lipase molecule is included. It would seem that fragment (336-449) is a functional domain of lipase.     

Effect of immunization to cholecystokinin fragment (30-33) on the behavior of albino rats

Active immunisation of albino rats by the BSA-conjugated CCK-4 induced formation of antibodies to the CCK-4 and some long-term changes of the rat behaviour. These changes were contrary to anxiogenic effect of the CCK-4 and demonstrated an anxiolytic effect of the immunisation. The data obtained suggest a possibility of an immunocorrection of pathological anxiety and fear by an inverse immunoregulation.     

Synthesis of the fragments A1-,, A9-15 and A16-21 of ovine insulin A chain using the S-tert-butylmercapto residue for thiol protection (author's transl)

The following paper describes the synthesis of the fragments A1-8, A9-15 and A16-21 of the ovine insulin A chain using the S-tert-butylmercapto residue for thiol protection. The synthesized fragments, which showed a good solubility in organic solvents, were partially deprotected with trifluoracetic acid and converted to the corresponding S-sulfonates quantitatively by oxidative sulfitolyses at pH 7.6.     

Effects of hind limb denervation on the development of appendicular ossicles in the Dwarf African Clawed Frog,Hymenochirus boettgeri (Anura: Pipidae)

Sesamoids and other appendicular ossicles are common in other classes of vertebrates but comparatively rare in amphibians. The pipid frog Hymenochirus boettgeri (Boulenger, G. A. 1899. On Hymenochirus, a new type of aglossal batrachians. – Annals of the Magazine of Natural History Series 7: 122–125) is unusual among anurans in having seven (or more) appendicular ossicles in each hind limb. Sesamoids are often associated with muscles and tendons, and their development is usually regarded as mediated by or correlated with function. This study investigated the effects of paralysis (loss of function) on development of ossicles in the hind limb of Hymenochirus. Complete denervation of the right sciatic nerve was performed at developmental stages 63 and 66, and the animals maintained for a further 6–7 or 12–13 weeks. Specimens were cleared and double stained for cartilage and bone. There were no gross morphological differences between control and sham operated groups. The lunulae were not affected by paralysis, whereas the fabella arose later and/or regressed in some specimens. The distal os sesamoides tarsalia (OST) was shorter in paralysed individuals, and both the distal OST and cartilagines plantares showed delayed maturation. Denervation of the hind limb thus affected the timing of appearance, maintenance and rate of maturation of some sesamoid bones in Hymenochirus, but had no effect on others.     

Effects of the beta(2)-agonist clenbuterol on respiratory and limb muscles of weaning rats

The aim of this study was to analyze the effects of chronic administration of the beta(2)-agonist clenbuterol (1.5 mg x kg(-1) x day(-1) for 4 wk in the drinking water) on respiratory (diaphragm and parasternal intercostal) and hindlimb (tibialis and soleus) muscles in young rats during postnatal development (21 to 49 postnatal days). The treatment resulted in very little stimulation of muscle growth. Significant slow-to-fast transitions in the expression of myosin heavy chain isoforms and significant increases in the myofibrillar ATPase activity were found in the diaphragm and soleus, whereas tibialis anterior and intercostal muscles did not show any significant fiber-type alteration. Decrease of oxidative enzyme activities and increase of glycolytic enzyme activities were also observed. It is concluded that whereas the growth stimulation is age dependent and only detectable in adult rats, the fiber-type transformation is also present in weaning rats and particularly evident in the soleus and diaphragm. The fiber-type transformation caused by clenbuterol might lead to an enhancement of contractile performance and also to a reduced resistance to fatigue.     

Effect of the corticoliberin fragment CRF(4-6) on blood pressure and heart rate in rats

The effects of tripeptide corticoliberin fragment CRF(4-6) (Pro-Pro-Ile) on blood pressure and heart rate of rats were investigated. Intracerebroventricularly injected CRF(4-6) (1.5-15.0 nmol/head) increases the mean arterial pressure and heart rate in conscious and anaesthetized animals in a dose-dependent manner. Corticotropin releasing factor antagonist: alpha-helical CRF(9-41) (6.5 nmol) completely abolished the influence of tripeptide CRF(4-6) (1.5 nmol) on blood pressure and heart rate in anaesthetized rats. This result indicates that corticoliberin receptors are involved in cardiovascular effects of CRF(4-6).     

Intracellular delivery of phosphatidylinositol (3,4,5)-trisphosphate causes incorporation of glucose transporter 4 into the plasma membrane of muscle and fat cells without increasing glucose uptake

Insulin stimulates glucose uptake into muscle and fat cells by translocating glucose transporter 4 (GLUT4) to the cell surface, with input from phosphatidylinositol (PI) 3-kinase and its downstream effector Akt/protein kinase B. Whether PI 3,4,5-trisphosphate (PI(3,4,5)P(3)) suffices to produce GLUT4 translocation is unknown. We used two strategies to deliver PI(3,4,5)P(3) intracellularly and two insulin-sensitive cell lines to examine Akt activation and GLUT4 translocation. In 3T3-L1 adipocytes, the acetoxymethyl ester of PI(3,4,5)P(3) caused GLUT4 migration to the cell periphery and increased the amount of plasma membrane-associated phospho-Akt and GLUT4. Intracellular delivery of PI(3,4,5)P(3) using polyamine carriers also induced translocation of myc-tagged GLUT4 to the surface of intact L6 myoblasts, demonstrating membrane insertion of the transporter. GLUT4 translocation caused by carrier-delivered PI(3,4,5)P(3) was not reproduced by carrier-PI 4,5-bisphosphate or carrier alone. Like insulin, carrier-mediated delivery of PI(3,4,5)P(3) elicited redistribution of perinuclear GLUT4 and Akt phosphorylation at the cell periphery. In contrast to its effect on GLUT4 mobilization, delivered PI(3,4,5)P(3) did not increase 2-deoxyglucose uptake in either L6GLUT4myc myoblasts or 3T3-L1 adipocytes. The ability of exogenously delivered PI(3,4,5)P(3) to augment plasma membrane GLUT4 content without increasing glucose uptake suggests that input at the level of PI 3-kinase suffices for GLUT4 translocation but is insufficient to stimulate glucose transport.