Ellger et al. Glucose control and regional NO metabolism

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Parenteral nutrition

The animals were fed by a total parenteral nutrition (TPN) composed according to the animals needs. We choose for total intravenous nutrition as this is the only way to assure equal nutrient intake of the rabbits. To account for different glucose needs in order to achieve the two levels of blood glucose in the 4 groups without exposing the animal to fluid overload, we composed two different PN solutions. Both assured equal fat and amino acid intake (each at 1.2 g/kg/d). For animals requiring less than 2 g glucose per hour to achieve target blood glucose, we used PN containing 35% Clinomel N7 (Baxter, Clinitec, Maurepas Cedex, France) and 65% Hartman solution. For animals requiring more than 2 g glucose per hour, we used PN containing 35% Clinomel, 35% Hartmann solution and 30% glucose 50%. Both solutions contained 3.22 g of arginine per litre. Intravenous infusions were prepared daily under sterile conditions and weighed before and after administration for exact quantification of intake.

NO-plasma-levels

NO levels in plasma were quantified by measurement of serum nitrate plus nitrite levels using nitrate reductase and the colorimetric Griess reaction. A 1:4 dilution in PBS of serum samples (200 µl) was ultrafiltered (6000 g, 45 min, 4°C) through 10,000 kDa molecular weight filters (Ultrafree-MC Biomax, Millipore, Billerica, MA, USA). 50 µl of the filtrates was used in duplicate. A standard curve of 50 µl 0-50 µM nitrate was included in each assay. Nitrate in the standards and samples was converted to nitrite by addition of 50 µl PBS with 600 U/l nitrate reductase from Aspergillus (Roche, Vilvoorde, Belgium) and 100 µM NADH. Complete conversion was obtained with an incubation time of 4 hours at room temperature, as evaluated by comparison of a converted nitrate standard curve with a nitrite standard curve. Nitrite was then measured after addition of 50 µl PBS, 50 µl 1% sulfanilamide in 5% phosphoric acid and 50 µl 0.1% N-(1-naphtyl)ethylenediamine dihydrochloride. The absorbance was read at 540 nm and corrected for absorbance at 650 nm.

Real-time PCRFor rtPCR the following combinations of Primers and Probes were used: eNOS (GenBank accession number AY179960): Forward primer 5’-ggcatcaccaggaagaagacc-3’, reverse primer 5’-tcactctctttgccatca-3’, TaqMan Probe 5’-agtgaagatctctgcctcactcat -3’; iNOS (GenBank accession number AF469048): Forward primer 5’-cccttcaacggctggtacat-3’, reverse primer 5’-gcgtctccagtcccatcct-3’, TaqMan Probe 5’-tgtgacgtccagcgctacaatatcctgg-3’;HPRT (GenBank accession number AF020294): Forward primer 5’-tgtagattttatcagactgaagagctactgt-3’, reverse primer 5’-aaggaaagcaaggtctgcattgtt-3’, TaqMan Probe 5’-tttccagttaaggttgagagatcatctccaccgat-3’;GTPCH (GenBank accession number NM00102407): Forward primer 5’- acgagatggtgattgtgaagga-3’, reverse primer 5’-aagataaccgatatgcacctttcc-3’, and TaqMan Probe 5’-ccatgtgtgagcatcatctggttccatt-3’.

NOS-activity

In tissue samples the conversion of L-3H-arginine to L-3H-citrulline was used as an index of NOS-activity. Tissue was homogenized in a buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, and 1 mM EGTA (pH 7.4) at a final concentration of 250 mg tissue/ml. Samples containing 100 µl of homogenate were incubated for 20 min with 100 µl incubationbuffer (40 mM HEPES pH 7.4, 1 mM reduced b-NADPH, 2 mM CaCl2, 24 µM L-arginine, 4.5 µCi L-2,3-3H-L-arginine (Perkin Elmer, Wellesley, MA, USA) at 37°C). Samples were processed in duplicate and incubated in the presence or absence of L-NAME (20 mM). The reaction was stopped by adding 1 ml ice cold stop reagent (10 mM EGTA, 1 mM citrulline, 100 mM PIPES, pH 5.5). The mixture was applied to a cation-binding Dowex W50 column and washed 3 times with MQ-water. In the eluate the counts per minute reflecting the amount of generated L-citrulline were measured. The difference of counts per minute with and without L-NAME was considered to mirror specific NOS-activity. Individual samples with a counts-variation greater than 20% were reanalyzed.

Endothelial function in aortic vascular rings

For evaluating endothelial function, we decided to examine NO-mediated endothelium-dependent vasodilatation in isolated perfused aortic rings since this method is most sensitive to detect endothelial dysfunction caused by alterations in the microenvironment.

Immediately after sacrificing the animal, thoracic aorta was carefully separated from connective tissue and 5 mm segments were sliced. Suspended by stainless steel clips they were immediately placed into an organ bath filled with modified Krebs solution (NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25.0, Na2CaEDTA 0.026, KH2PO4 1.2 (in mmol/L), Glucose 5.5 mmol/L, Insulin 40 mU/L, Indometacin 10-5 mol/l, bubbled with 95%O2/ 5%CO2, pH 7.4 ±0.05, 37.4°C). To exclude confounding effects exerted by the glucose and insulin content in the organ bath, the tests were run in parallel in a modified organ bath with glucose and insulin concentrations reflecting the animals` randomisation (“high insulin solution”: insulin 100 mU/l, “high glucose solution”: glucose 16.5 mmol/L). Supplementary, we conducted the experiment in healthy animals in organ baths containing glucose and insulin reflecting the 4 experimental groups. We did not control for osmolality in the different organ baths as it is known that altered osmolality does not affect endothelium-dependent and independent vasorelaxation. Connected to a force transducer (F30, HSE, March-Hugstetten, Germany) isometric tension was recorded continuously. After one hour of stabilisation, pretension was adjusted to 2 g. Subsequently, Norephedrine (NE) was added to the organ bath to a concentration of 10-6 mol/l. When reaching steady contraction state, changes in contraction were recorded in response to drugs added to the organ bath: 1) Acetylcholine (Ach) in stepwise cumulative doses of 10 nmol/l to 10 µmol/l. As Ach induces NO release from NO-synthetase (NOS)-activity, it marks endothelium dependent vasorelaxation and thus endothelial function. Next dose was only administered after retaining stable conditions, 2) Ach as described above in an organ bath supplemented with L-nitro-arginine-methyl-ester (L-NAME, 10-4 mol/l), a nonspecific NOS-blocker to exclude endothelium independent actions of Ach, and 3) Sodium Nitroprusside (Nipruss, Sodium-Nitroferricyanide(III)dihydrate) to cumulative doses of 10 nmol/l to10 µmol/l to assess NO-mediated endothelium independent vasorelaxation. After each step, the organ bath was flushed three times and pretension was readjusted, the system was allowed to equilibrate for 30 minutes (all chemicals from Sigma Aldrich, Bornem, Belgium). Relaxation of the rings was expressed as percent of developed tension induced by NE. Measurements in sick animals were compared to healthy controls.

Measurements in organ bath supplemented with L-NAME revealed no relaxation to Ach whereas the relaxation to Nipruss was unaffected in all groups. This suggests that endothelium independent vasorelaxation was intact in all animals and that the reduced vasorelaxation in response to Ach in hyperglycemic groups is an effect of reduced endothelium derived NO-release. Measurements in organ baths containing different concentrations of insulin or glucose, respectively, revealed no differences in vasorelaxation excluding major confounding effects of the composition of the organ bath.

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