Metabolic and physiological studies in a rat model of type 2 diabetes

Wijekoon, Enoka P. (2006) Metabolic and physiological studies in a rat model of type 2 diabetes. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Mild hyperhomocysteinemia is a risk factor for many diseases, including cardiovascular disease. We determined the effects of insulin resistance and of Type 2 diabetes on homocysteine and amino acid metabolism using Zucker diabetic fatty rats (ZDF/Gmi-fa/fa and ZDF/Gmi +/?). Plasma total homocysteine was reduced in ZDF fa/fa rats by 24% in the pre-diabetic insulin-resistant stage while in the frank diabetic stage there was a 59% reduction. Hepatic activities of several enzymes that remove homocysteine: cystathionine β-synthase, cystathionine γ-lyase and betaine:homocysteine methyltransferase were increased as well as methionine adenosyltransferase. Cystathionine β-synthase and betaine:homocysteine methyltransferase mRNA levels and the hepatic level of S-adenosylmethionine were also increased in the ZDF fa/fa rats. Studies with primary hepatocytes showed that homocysteine export and the transsulfuration flux in cells from ZDF fa/fa rats were particularly sensitive to the presence of betaine, which reduced these fluxes. Interestingly, the liver betaine concentration was found to be significantly lower in the ZDF fa/fa rats at both 5 and 11 weeks. We conclude that the decreased insulin action and the elevation of the counter-regulatory hormones observed in diabetes may be responsible for the more efficient homocysteine removal seen in the ZDF fa/fa rats. -- Amino acids were measured in plasma, liver and skeletal muscle and the ratios of plasma: liver and plasma: skeletal muscle, were calculated (Chapter 4). At the insulin-resistant stage, the plasma concentrations of the gluconeogenic amino acids aspartate, serine, glutamine, glycine and histidine were decreased in the ZDF fa/fa rats while taurine, α-aminoadipic acid, methionine, phenylalanine, tryptophan and the three branched-chain amino acids were significantly increased. At the diabetic stage, a larger number of gluconeogenic amino acids showed decreased plasma concentrations. The three branched chain amino acids again showed elevated plasma concentrations. In the liver and the skeletal muscles, many of the gluconeogenic amino acids showed lowered concentrations at both stages while the levels of one or all of the branched-chain amino acids were elevated. These changes in amino acid concentration are similar to changes seen in Type 1 diabetes. However, it is evident that insulin resistance alone is capable of bringing about many of the changes in amino acid metabolism observed in Type 2 diabetes. -- The next part of the study focused on the effects of the insulin-sensitizing drug, Rosiglitazone, on homocysteine metabolism (Chapter 5). Rosiglitazone is an agonist of the peroxisome proliferator activated receptor y and is used as an anti-diabetic agent to treat Type 2 diabetes. Male ZDF fa/fa and ZDF fa/+ rats, aged 6 weeks were each divided into 2 groups: Rosiglitazone-treated (RSG) and untreated, and were killed at 12 weeks of age. Rosiglitazone treatment was able to maintain a normal plasma glucose level in the ZDF fa/fa (RSG) rats. At 12 weeks of age, ZDF fa/fa (untreated) rats developed type 2 diabetes as indicated by a 3-fold increase in plasma glucose, while plasma insulin level was similar to ZDF fa/+ rats. The significant reduction observed in plasma homocysteine in the ZDF fa/fa (untreated) rats was returned towards normal by Rosiglitazone treatment. The elevated activity of the transsulfuration enzyme, cystathionine y-lyase, in the ZDF fa/fa (untreated) rats was corrected by Rosiglitazone treatment while cystathionine β-synthase was unaffected. The elevated activity of betaine:homocysteine methyltransferase observed in the ZDF fa/fa (untreated) rats was further increased by Rosiglitazone treatment. That the increased activity of betaine:homocysteine methyltransferase in the ZDF fa/fa (RSG) rats may be a function of increased lipid output from the liver in the face of the lipid redistribution seen with Rosiglitazone is supported by the changes that were observed in phospholipid metabolism. -- Type 1 and Type 2 diabetes as well as insulin resistance prior to the development of Type 2 diabetes have been reported to cause endothelial dysfunction. Hyperglycemia has been suggested to play a major role in this development of endothelial dysfunction. Hyperhomocysteinemia has also been identified as a causative agent of endothelial dysfunction. Our earlier studies (Chapter 3 and 5) showed plasma homocysteine to be lower in both insulin resistance and during Type 2 diabetes in ZDF fa/fa rats. We, therefore, designed a study to determine the effects of increased glucose and decreased homocysteine on the function of mesenteric arteries and the effects of normalization of these two parameters with the treatment of Rosiglitazone. 6 week old ZDF fa/+ and ZDF fa/fa rats were randomly assigned to RSG treated and untreated groups as in Chapter 3, and were killed at 6, 12 and 18 weeks of age. -- The endothelium dependent vasorelaxation in response to acetylcholine, the proteinase-activated receptor-2 (PAR2) agonist 2-furoyl-LIGRLO-amide (2fli) and arachidonic acid were preserved in the ZDF fa/fa mesenteric arteries despite the hyperinsulinemia at 6 weeks and the hyperglycemia at 12 and 18 weeks. Despite the normalization of plasma glucose and near normalization of plasma homocysteine with the treatment of Rosiglitazone the endothelium dependent vasorelaxation in response to the above agonists did not change, reflecting the lack of any endothelial defects in the mesenteric arteries of these rats. -- The inability of the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) to completely abolish vasorelaxation in response to both acetylcholine and 2fli demonstrates the important role played by endothelium-derived-hyperpolarizing factor in the relaxation of small caliber blood vessels in response to these 2 agonists. Studies conducted with inhibitors of cyclo-oxygenase 112 (COX1/2) and calcium activated potassium channels (Kca) to identify the possible nitric oxide synthase independent mechanisms of vasorelaxation indicated both COX and Kca channels to be involved. Kca channels however, were shown to play a greater role than COX 112 as previously reported. -- We also observed an increased sensitivity towards the P AR2 agonist 2fli by the ZDF fa/fa rat vessels. This may be consistent with the increase in pro-inflammatory substances in the ZDF rat model, as P AR2 expression has been shown to increase during stress to the blood vessels and in the presence of various cytokines and pro-inflammatory substances. -- Our data also did not indicate a reduction in vascular smooth muscle cell responsiveness to nitric oxide in the ZDF fa/fa rats compared to ZDF fa/+ rats. Both groups of rats however, displayed a reduced relaxation response to sodium Nitroprusside compared to previously reported values. Treatment with Rosiglitazone was able to successfully restore this responsiveness in both groups of rats. A similar increase in responsiveness was also seen in the ZDF fa!+ and ZDF fa/fa untreated rat vessels as well as ZDF fa/fa (RSG) vessels incubated with L-NAME. -- The normal endothelium dependent vasorelaxation displayed by the ZDF fa/fa rats despite the hyperglycemia indicates that hyperglycemia alone may not be able to cause endothelial dysfunction. Since plasma homocysteine levels in these rats were decreased, it leaves us with the interesting possibility that homocysteine may act as a balance point for development of endothelial dysfunction in this model of Type 2 diabetes.

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