Increased glycation and oxidative damage to apolipoprotein B100 of LDL cholesterol in patients with type 2 diabetes and effect of metformin

OBJECTIVE The aim of this study was to investigate whether apolipoprotein B100 of LDL suffers increased damage by glycation, oxidation, and nitration in patients with type 2 diabetes, including patients receiving metformin therapy. RESEARCH DESIGN AND METHODS For this study, 32 type 2 diabetic patients and 21 healthy control subjects were recruited; 13 diabetic patients were receiving metformin therapy (median dose: 1.50 g/day). LDL was isolated from venous plasma by ultracentrifugation, delipidated, digested, and analyzed for protein glycation, oxidation, and nitration adducts by stable isotopic dilution analysis tandem mass spectrometry. RESULTS Advanced glycation end product (AGE) content of apolipoprotein B100 of LDL from type 2 diabetic patients was higher than from healthy subjects: arginine-derived AGE, 15.8 vs. 5.3 mol% (P < 0.001); and lysine-derived AGE, 2.5 vs. 1.5 mol% (P < 0.05). Oxidative damage, mainly methionine sulfoxide residues, was also increased: 2.5 vs. 1.1 molar equivalents (P < 0.001). 3-Nitrotyrosine content was decreased: 0.04 vs. 0.12 mol% (P < 0.05). In diabetic patients receiving metformin therapy, arginine-derived AGE and methionine sulfoxide were lower than in patients not receiving metformin: 19.3 vs. 8.9 mol% (P < 0.01) and 2.9 vs. 1.9 mol% (P < 0.05), respectively; 3-nitrotyrosine content was higher: 0.10 vs. 0.03 mol% (P < 0.05). Fructosyl-lysine residue content correlated positively with fasting plasma glucose. Arginine-derived AGE residue contents were intercorrelated and also correlated positively with methionine sulfoxide. CONCLUSIONS Patients with type 2 diabetes had increased arginine-derived AGEs and oxidative damage in apolipoprotein B100 of LDL. This was lower in patients receiving metformin therapy, which may contribute to decreased oxidative damage, atherogenicity, and cardiovascular disease

Visfatin is regulated by rosiglitazone in type 2 diabetes mellitus and influenced by NFκB and JNK in human abdominal subcutaneous adipocytes

Visfatin has been proposed as an insulin-mimicking adipocytokine, predominantly secreted from adipose tissue and correlated with obesity. However, recent studies suggest visfatin may act as a proinflammatory cytokine. Our studies sought to determine the significance of this adipocytokine and its potential role in the pathogenesis of T2DM. Firstly, we examined the effects of diabetic status on circulating visfatin levels, and several other adipocytokines, demonstrating that diabetic status increased visfatin*, TNF-α*** and IL-6*** compared with non-diabetic subjects (*p<0.05, **p<0.01, ***p<0.001, respectively). We then assessed the effects of an insulin sensitizer, rosiglitazone (RSG), in treatment naïve T2DM subjects, on circulating visfatin levels. Our findings showed that visfatin was reduced post-RSG treatment [vs. pre-treatment (*p<0.05)] accompanied by a reduction in HOMA-IR**, thus implicating a role for insulin in visfatin regulation. Further studies addressed the intracellular mechanisms by which visfatin may be regulated, and may exert pro-inflammatory effects, in human abdominal subcutaneous (Abd Sc) adipocytes. Following insulin (Ins) and RSG treatment, our in vitro findings highlighted that insulin (100 nM), alone, upregulated visfatin protein expression whereas, in combination with RSG (10 nM), it reduced visfatin*, IKKβ** and p-JNK1/2*. Furthermore, inhibition of JNK protein exacted a significant reduction in visfatin expression (**p<0.01), whilst NF-κB blockade increased visfatin (*p<0.05), thus identifying JNK as the more influential factor in visfatin regulation. Additional in vitro analysis on adipokines regulating visfatin showed that only Abd Sc adipocytes treated with recombinant human (rh)IL-6 increased visfatin protein (*p<0.05), whilst rh visfatin treatment, itself, had no influence on TNF-α, IL-6 or resistin secretion from Sc adipocytes. These data highlight visfatin's regulation by insulin and RSG, potentially acting through NF-κB and JNK mechanisms, with only rh IL-6 modestly affecting visfatin regulation. Taken together, these findings suggest that visfatin may represent a pro-inflammatory cytokine that is influenced by insulin/insulin sensitivity via the NF-κB and JNK pathways

A–E. Photographs of Immunological Staining of Visfatin Protein.

<p>This figure shows the immunological staining of visfatin in (A) human kidney negative (primary antibody omitted); (B) human kidney positive control; (C) human AT PBEF negative (primary antibody omitted); (D) human Om AT; (E) human Abd Sc AT. Red arrows highlight areas of red staining to show the presence of the visfatin protein.</p

The Effects of JNK and NF-κB Inhibition on Visfatin Protein Expression in Human Adipocytes.

<p>This figure shows the effect of JNK and NF-κB blockade, alone and in combination, on the mean relative fold protein expression (±SEM) of visfatin in isolated Abd Sc adipocytes compared with control (untreated adipocytes), given an arbitrary value of one. The representative Western blots are shown above (n = 4, p-values: p<0.05*, p<0.01**).</p

Clinical and Biochemical Characteristics of T2DM Subjects Compared with Control Subjects.

<p>Data are presented as mean (±SD), unless otherwise stated. Non-parametric data have been log transformed in which case they are presented as mean (interquartile range).</p

Visfatin Protein Expression in Relation to Adiposity and Depot Specificity.

<p>This figure shows the mean relative fold protein expression of visfatin in omental (Om) versus abdominal (Abd) adipose tissue depots in matched pairs, from a cohort of lean and obese subjects, where the Abd Sc depot has been standardized. The representative Western blots are shown above. Lean samples: n = 7 & Obese samples: n = 6; p-values: p<0.001.</p