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Effect of heart work and insulin on the incorporation of [14C]glucose into hexose phosphates, uridine diphosphate glucose and glycogen in the normal and insulin-deficient perfused rat heart under working and non-working conditions.

ABSTRACT: 1. The specific radioactivities of glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, UDP-glucose and glycogen, derived from [14C]gluocose, were determined in the normal and insulin-deficient (streptozotocin-diabetic and anti-insulin-serum-treated) perfused non-working and working rat heart. 2. The specific radioactivities of all glucose metabolities reached a plateau after about 10 min, except that for glycogen, which increased slightly but steadily over the whole observation period of 30min. 3. The specific radio-activities of fructose 6-phosphate, UDP-glucose and glycogen were slignificantly lower in the streptozotocin-diabetic heart than in the normal heart. 4. Mechanical work in the normal rat heart increased the specific radioactivities of glucose 1-phosphate, UDP-glucose and glycogen, but had little or no effect on those of gluose 6-phosphate and fructose 6-phosphate. 5. In the normal heart insulin strongly increased the specific radioactivities of all gluocse metabolites under all conditions tested. The maximum values achieved in the normal working heart in the presence of insulin were only about 15-20% above those in the normal non-working heart in the presence of insulin for the phosphorylated intermediates and about 40% above for glycogen. 6. In the streptozotocin-diabetic heart, work restored the specific radioactivities of all glucose metabolities to about normal values. 7. In the streptozotocin-diabetic heart insulin strongly increased the specific radioactivities of the direct glycogen precursors glucose 1-phosphate and UDP-glucose; the effect of insulin on glucose 6-phosphate and fructose 6-phosphate was less marked. These results confirm previous findings that the primary metabolic lesion in diabetic heart muscle is a defect of glycogen synthesis. The specific radioactivity of glycogen itself was increased sixfold. 8. Under all conditions tested the specific radioactivity of glucose 1-phosphate was always found to be higher than that of glucose 6-phosphate. This indicated either compartmentation of a small but metabolically very active pool of glucose 6-phosphate, or the existence of a hitherto unknown pathway of metabolism in which glucose 1-phosphate is the primary reaction product. For a number of reasons the authors prefer the first explanation, which could also account for the observation that in the perfused normal working and non-working heart the specific radioactivity of fructose 6-phosphate was always found to be higher than that of glucose 6-phosphate. This difference disappeared or was reversed in the rat hearts rendered insulin-insufficent by either streptozotocin or anti-insulin treatment.

SUBMITTER: Das I

PROVIDER: S-EPMC1172780 | biostudies-other | 1976 Mar

REPOSITORIES: biostudies-other

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Project description:We investigated the isolated working rat heart as a model to study early transcriptional remodeling induced by cardiac surgery with cardiopulmonary bypass (CPB). Because the mechanisms linking stress hyperglycemia and insulin resistance to increased peri-operative morbidity and mortality in the setting of open-heart surgery are still unclear, we also used this model to determine the transcriptional effects of exogenous glucose supply in absence or in presence of a preexisting state of myocardial insulin resistance. Male Sprague Dawley rats (200-224g) were kept on chow diet for 8 to 10 weeks, or fed a high-sucrose diet to induce myocardial insulin resistance. Following the feeding period, hearts were recovered, arrested in ice-cold Krebs-Henseleit (KH) buffer and subjected to 60 minutes normothermic reperfusion in the working mode with KH buffer supplemented with non-carbohydrate substrates plus glucose (25 mM) or mannitol (25 mM; osmotic control). Compared to non-perfused hearts, perfused hearts from chow-fed rats displayed a more than twofold increased expression for 71 genes (mannitol group) and 103 genes (glucose group) connected to inflammation, cell proliferation, and apoptosis. The same biological pathways were activated in hearts from insulin resistant rats, with 109 genes (mannitol group) and 70 genes (glucose group) up-regulated more than twofold when compared to the non-perfused hearts from high-sucrose diet-fed rats. The changes were highly similar to gene alterations occurring in the right atrium and left ventricle of open heart surgery patients, and included the up-regulation of the three master regulators of metabolic reprogramming MYC, NR4A1 and NR4A2. Targeted pathway analysis revealed an up-regulation of metabolic processes associated with the proliferation and activation of tissue-resident macrophages and fibroblasts. Although the remodeling occurred independently from glucose, glucose further increased the expression of a subset of genes associated with polarization of tissue reparative M2 macrophages. However, glucose failed to enhance the expression of M2-related markers in the isolated heart from rats rendered insulin resistant by high-sucrose feeding. These results expose the cellular components of the heart as a significant source of proinflammatory mediators released in response to stress associated with cardiac surgery with CPB, and suggest a major role for glucose as a signal in the determination of resident cardiac macrophage polarization.

Project description:1. In incubation mixtures containing digitonin-activated or untreated preparations from rat liver, albumin-solubilized bilirubin as the acceptor substrate and (a) UDP-glucuronic acid, (b) UDP-glucose or (c) UDP-xylose as the sugar donor, formation of the following ester glycosides was demonstrated: with (a), bilirubin beta-d-monoglucuronoside, with (b), bilirubin beta-d-monoglucoside and with (c), bilirubin monoxyloside or mixtures of the mono-and di-xyloside. 2. With UDP-glucuronic acid prolonged incubation and variation of the composition of the incubation mixtures yielded equimolar amounts of azodipyrrole (I) and azodipyrrole beta-d-monoglucuronoside (II) after treatment of the incubation mixtures with the diazonium salt of ethyl anthranilate. The azo-derivatives were identified by t.l.c. by reference to known compounds and by the following chemical tests. After ammonolysis the conjugated azo-derivative (II) yielded d-glucuronic acid and the carboxylic acid amide of azodipyrrole, indicating transfer of a glucuronic acid residue to the carboxylic acid groups of bilirubin. The beta-d-configuration of the sugar moiety and binding at C-1 were demonstrated by enzymic hydrolysis tests. 3. Analogous evidence established the structure of the reaction product obtained with UDP-glucose as the sugar donor, as bilirubin beta-d-monoglucoside. 4. With UDP-xylose as the sugar donor xylosyl transfer to the carboxylic acid groups of bilirubin with attachment at C-1 was demonstrated in an analogous way. A beta-d-configuration is considered very likely, but requires confirmation. 5. Monoxyloside formation was predominant at pH7.4, whereas at decreasing pH values increasing fractions of the substrate were converted into the dixyloside. Prolonged incubation, low concentrations of bilirubin and high concentrations of UDP-xylose favoured diconjugate formation. The available evidence supports the synthesis sequence: bilirubin --> bilirubin monoxyloside --> bilirubin dixyloside.

Project description:BACKGROUND: Uridine diphosphate (UDP) is an extracellular nucleotide signaling molecule implicated in diverse biological processes via specific activation of pyrimidinergic receptor P2Y, G Protein-Coupled, 6 (P2Y6). There is very little knowledge about the function and mechanism of UDP in rheumatoid arthritis (RA). METHODS: This study used a quasi-targeted liquid chromatography-mass spectrometry (LC-MS) approach to investigate the unique expression of metabolites in RA synovial fluids (SF) (n = 10) with samples from osteoarthritis (OA) as controls (n = 10). RA fibroblast-like synoviocytes (FLSs) were collected from synovial tissues (n = 5) and cultured with UDP or MRS2578, a P2Y6 antagonist, and FLSs from OA were used as controls (n = 5). Rats with collagen-induced arthritis (CIA) were injected with UDP, MRS2578 or both (n = 9 for each group). P2Y6 expression was examined using real-time PCR, Western blotting and immunohistochemistry. Cell proliferation, apoptosis and migration of RA FLSs were measured using CCK-8 assay, real-time cell analysis, flow cytometry, wound healing assay and Transwell assay, respectively. The UDP levels in the culture medium, synovial fluid (n = 36) and peripheral blood (n = 36) of RA and CIA rats were measured using a Transcreener UDP Assay. Levels of proinflammatory cytokines were measured using a flow assay. Interleukin-6 (IL-6) levels were measured using ELISA and flow. RESULTS: LC-MS analysis detected significantly increased UDP levels in RA SF compared with OA SF, and the level was positively correlated with anticyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF)levels in RA. The increased UDP concentration was verified in the blood and synovial fluids of RA patients compared with samples from OA patients and healthy volunteers, respectively. UDP stimulated cell proliferation, migration and IL-6 secretion in RA FLSs and inhibited their apoptosis in culture, and MRS2578 inhibited these effects of UDP. UDP injection accelerated CIA and stimulated IL-6 production rather than other proinflammatory cytokines in the rat model, but simultaneous injection of MRS2578 suppressed these effects and alleviated CIA. P2Y6 expression was increased in RA and CIA synovial tissues. CONCLUSION: These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.

Project description:1. Digitonin-treated and untreated homogenates, cell extracts and washed microsomal preparations from liver of Wistar R rats are capable of transferring sugar from UDP-glucose or UDP-xylose to bilirubin. No formation of bilirubin glycosides occurred with UDP-galactose or d-glucose, d-xylose or d-glucuronic acid as the sources of sugar. 2. Procedures to assay digitonin-activated and unactivated bilirubin UDP-glucosyltransferase and bilirubin UDP-xylosyltransferase were developed. 3. In digitonin-activated microsomal preparations the transferring enzymes had the following properties. Both enzyme activities were increased 2.5-fold by pretreatment with digitonin. They were optimum at pH6.6-7.2. Michaelis-Menten kinetics were followed with respect to UDP-glucose. In contrast, double-reciprocal plots of enzyme activity against the concentration of UDP-xylose showed two intersecting straight-line sections corresponding to concentration ranges where either bilirubin monoxyloside was formed (at low UDP-xylose concentrations) or where mixtures of both the mono- and di-xyloside were synthesized (at high UDP-xylose concentrations). Both enzyme activities were stimulated by Mg(2+); Ca(2+) was slightly less, and Mn(2+) slightly more, stimulatory than Mg(2+). Of the activities found in standard assay systems containing Mg(2+), 58-78% (substrate UDP-glucose) and 0-38% (substrate UDP-xylose) were independent of added bivalent metal ion. Double-reciprocal plots of the Mg(2+)-dependent activities against the concentration of added Mg(2+) were linear. 4. In comparative experiments the relative activities of liver homogenates obtained with UDP-glucuronic acid, UDP-glucose and UDP-xylose were 1:1.5:2.7 for untreated preparations and 1:0.29:0.44 after activation with digitonin. 5. Bilirubin UDP-glucuronyltransferase was protected against denaturation by human serum albumin, whereas bilirubin UDP-xylosyltransferase was not. 6. Digitonin-treated and untreated liver homogenates from Gunn rats were inactive in transferring sugar to bilirubin from UDP-glucuronic acid (in agreement with the work of others), UDP-glucose or UDP-xylose.

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Effect of heart work and insulin on the incorporation of [14C]glucose into hexose phosphates, uridine diphosphate glucose and glycogen in the normal and insulin-deficient perfused rat heart under working and non-working conditions.

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