Abstract
BackgroundAsymmetric NG,NG-dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is regulated by the enzymatic participants of synthetic and metabolic processes, i.e., type I protein N-arginine methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH). Previous reports have demonstrated that circulating ADMA levels can vary in patients with type 1 and type 2 diabetes mellitus (T2DM). White adipose tissue expresses the full enzymatic machinery necessary for ADMA production and metabolism; however, modulation of the activities of adipose ADMA-related enzymes in T2DM remains to be determined.MethodsA rodent model of T2DM using 11- and 20-week old Goto-Kakizaki (GK) rats was used. The expression and catalytic activity of PRMT1 and DDAH1 and 2 in the white adipose tissues (periepididymal, visceral and subcutaneous fats) and femur skeletal muscle tissue were determined by immunoblotting, in vitro methyltransferase and in vitro citrulline assays.ResultsNon-obese diabetic GK rats showed low expression and activity of adipose PRMT1 compared to age-matched Wistar controls. Adipose tissues from the periepididymal, visceral and subcutaneous fats of GK rats had high DDAH1 expression and total DDAH activity, whereas the DDAH2 expression was lowered below the control value. This dynamic of ADMA-related enzymes in white adipose tissues was distinct from that of skeletal muscle tissue. GK rats had lower levels of serum non-esterified fatty acids (NEFA) and triglycerides (TG) than the control rats. In all subjects the adipose PRMT1 and DDAH activities were statistically correlated with the levels of serum NEFA and TG.ConclusionActivities of PRMT1 and DDAH in white adipose tissues were altered in diabetic GK rats in an organ-specific manner, which was reflected in the serum levels of NEFA and TG. Changes in adipose ADMA-related enzymes might play a part in the function of white adipose tissue.
Highlights
Asymmetric NG,NG-dimethylarginine (ADMA), a naturally occurring inhibitor of nitric oxide synthase (NOS), is produced by the proteolysis of intracellular proteins that are posttranslationally modified by type I protein N-arginine methyltransferase (PRMT) [1]
Based upon these characteristics of GK rats, the present study aims to explore the possible involvement of PRMT1 and Dimethylarginine dimethylaminohydrolase 1 and 2 (DDAH1/2) in the function of white adipose tissue through the dynamics and characteristic of these ADMA-related enzymes in type 2 diabetes mellitus (T2DM)
The PRMT1 protein level of periepididymal fat decreased by 12% in 11-week-old GK rats compared to that of the agematched Wistar controls (p = 0.037) (Figure 1A)
Summary
Animals Both 11- and 20-week-old male Wistar and GK rats were housed with free access to water and standard rat diet (352 kcal/100 g) and measured for body-weight. The tissue extracts were resolved using 3 × SDS-polyacrylamide gel electrophoresis (PAGE) buffer (187.5 mM Tris–HCl, 6% SDS, 30% glycerol, 150 mM dithiothreitol, 0.3% bromophenol blue, pH 6.8), and the samples were boiled for 5 min at 95°C. The proteins in the gel were transferred to a nitrocellulose membrane (GE Healthcare Biosciences, Tokyo, Japan) by electroblotting. The supernatants were subjected to SDS-PAGE in a 5% Tris–HCl gel, transferred to a poly(vinylidene difluoride) membrane (Millipore, Billerica, MA), sprayed with En3hance (PerkinElmer Life and Analytical Sciences, Boston, MA), and exposed to Kodak BioMax MS film (Eastman Kodak Company, Rochester, NY) with the Transcreen LE Intensifying Screen (Eastman Kodak Company) for 2–5 days at −80°C. Tissue extracts (300 μg) were incubated with urease (100 U/ml; Sigma-Aldrich) for 15 min at 37°C, and 400 μl of ADMA (1 mM; Alexis Biochemicals, Plymouth Metting, PA) in sodium phosphate buffer was added to 100 μl of each sample for 45 min at 37°C.
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