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.Cardiovascular disease (CVD) is the major cause of premature death in diabetes. Type 2 diabetes is associated with a twofold to threefold increased risk of coronary heart disease in men and a threefold to fivefold increased risk in women, relative to the nondiabetic population (1). Dyslipidemia is a key feature of diabetic CVD where small dense LDL particles pose a major atherogenic threat. The underlying mechanism producing small, dense LDL is related to hepatic oversecretion of apolipoprotein B100 (apoB100) and impaired clearance of LDL by the high-affinity LDL receptor in which both hepatic and peripheral tissues participate (2). The normal residence time of LDL in plasma is 3 days but this is increased to 5 days for small, dense, highly atherogenic LDL (3). Atherogenicity and plasma residence time of LDL may be influenced by damage to apoB100 by glycation, oxidation, and nitration but the quantitative amounts of damage in healthy human subjects and diabetic patients remain unclear.Glycation of proteins is a complex series of parallel and sequential reactions collectively called the Maillard reaction. Early stage reactions are directed to lysine and NH₂-terminal amino acid residues leading to the formation of the early glycation adduct, fructosyl-lysine (FL), and other fructosamine derivatives. Later stage reactions form advanced glycation end products (AGEs). FL degrades slowly to form AGEs. Glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG) are physiological dicarbonyl metabolites and potent glycating agents formed by the degradation of glycolytic intermediates, glycated proteins, and lipid peroxidation. They react with proteins to form AGEs directed mainly to arginine residues—often functionally important arginine residues. The most important AGEs quantitatively are hydroimidazolones derived from arginine residues modified by glyoxal, methylglyoxal, and 3-DG: G-H1, MG-H1, and 3DG-H, respectively. N_ω-carboxymethyl-arginine (CMA) is a further arginine-derived adduct formed by glyoxal. Other important and widely studied AGEs are N_ε-carboxymethyl-lysine (CML), N_ε-carboxyethyl-lysine (CEL), and pentosidine. Markers of oxidative damage to proteins are methionine sulfoxide (MetSO), formed by the oxidation of methionine, and dityrosine, formed by oxidative cross-linking of tyrosine. A widely studied marker of nitration damage to proteins is 3-nitrotyrosine (3-NT) (rev. in 4) (Fig. 1).Open in a separate windowFIG. 1.Molecular structures of protein glycation, oxidation, and nitration residues.Metformin is the most widely prescribed oral glucose-lowering agent for the treatment of type 2 diabetes. It improves glycemic control and decreases the risk of CVD (5). Metformin therapy of type 2 diabetic patients increased LDL particle size (6) and decreased plasma concentrations of remnant lipoprotein cholesterol, a predictor of myocardial infarction and thought to reflect increased residence time and atherogenicity of cholesterol ester–rich chylomicrons and VLDL (7). Metformin also decreased the plasma concentrations of methylglyoxal in diabetic patients (8) and may decrease oxidative stress and related oxidation of LDL (9).In this study, we used the gold standard method of stable isotopic dilution analysis liquid chromatography–tandem mass spectrometry (LC-MS/MS) to measure protein glycation, oxidation, and nitration adducts in apoB100 of LDL to assess whether there is increased lipoprotein damage in patients with type 2 diabetes with respect to normal healthy control subjects and to investigate the effect of metformin therapy.     

The depth of the prostatic apex is an independent predictor of positive apical margins at radical prostatectomy

Study Type – Therapy (case series)
Level of Evidence 4

OBJECTIVE

To determine the effect of a deep and narrow pelvis on apical positive surgical margins (PSM) at radical prostatectomy (RP), controlling for other clinical and pathological variables and surgical approach, i.e. open retropubic (RRP) vs laparoscopic (LRP), as apical dissection is expected to be more challenging at RP with a prostate situated deep in a narrow pelvis.

PATIENTS AND METHODS

From July 2003 to January 2005, 512 consecutive patients with preoperative prostate magnetic resonance imaging (MRI) underwent RRP or LRP with no previous radio‐ or hormonal therapy. An additional 74 patients with preoperative MRI undergoing RP from December 2001 to June 2007 who had an apical PSM were also included, with 586 patients comprising the study population. Bony and soft‐tissue pelvic dimensions, including interspinous distance (ISD), bony (BFW) and soft tissue (SW) pelvic width, apical prostate depth (AD) and symphysis pubis angle, were measured on preoperative MRI. The pelvic dimension index (PDI), bony width index (BWI) and soft‐tissue width index (SWI) were defined as ISD/AD, BFW/AD and SW/AD, respectively. Multivariate logistic regression was used to assess the effect of pelvic dimensions on apical PSM, controlling for surgical approach and clinical and pathological variables.

RESULTS

There was no significant difference in ISD, BFW, SW or symphysis angle between patients with and without apical PSM. The AD was significantly greater in men with an apical PSM and consequently PDI, BWI and SWI were significantly lower in men with an apical PSM. Each of PDI, AD, BWI and SWI was a significant independent predictor of apical PSM, independent of surgical approach, and other clinicopathological variables. The main limitations of the study were that it was retrospective, and the relatively few patients with apical PSM.

CONCLUSIONS

Apical prostate depth is an independent risk factor for apical PSM at RP. MRI pelvimetry might allow for preoperative planning of the approach to RP.     

Role of Oxidative Stress in a Rat Model of Radiation-Induced Erectile Dysfunction

IntroductionChronic oxidative stress is one of the major factors playing an important role in radiation-induced normal tissue injury. However, the role of oxidative stress in radiation-induced erectile dysfunction (ED) has not been fully investigated.AimsTo investigate role of oxidative stress after prostate-confined irradiation in a rat model of radiation-induced ED.MethodsFifty-four young adult male rats (10–12 weeks of age) were divided into age-matched sham radiotherapy (RT) and RT groups. Irradiated animals received prostate-confined radiation in a single 20 Gy fraction.Main Outcome MeasuresIntracavernous pressure (ICP) measurements with cavernous nerve electrical stimulation were conducted at 2, 4, and 9 weeks following RT. The protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits (Nox4 and gp91^phox), markers of oxidative DNA damage (8-hydroxy-2′-deoxyguanosine [8-OHdG]), lipid peroxidation (4-hydroxynonenal [4HNE]), and inflammatory response including inducible nitric oxide synthase, macrophage activation (ED-1), and nitrotyrosine, and endogenous antioxidant defense by nuclear factor erythroid 2-related factor (Nrf2) were evaluated in irradiated prostate tissue and corpora cavernosa (CC). In addition, we investigated the relationships between results of ICP/mean arterial pressure (MAP) ratios and expression level of oxidative stress markers.ResultsIn the RT group, hemodynamic functional studies demonstrated a significant time-dependent decrease in ICP. Increased expression of Nox4, gp91^phox, 8-OHdG, and 4HNE were observed in the prostate and CC after RT. Similarly, expressions of inflammatory markers were significantly increased. There was a trend for increased Nrf2 after 4 weeks. ICP/MAP ratio negatively correlated with higher expression level of oxidative markers.ConclusionsNADPH oxidase activation and chronic oxidative stress were observed in irradiated prostate tissue and CC, which correlated with lower ICP/MAP ratio. Persistent inflammatory responses were also found in both tissues after RT. These findings suggest that oxidative stress plays a crucial role in the development of radiation-induced ED. Kimura M, Rabbani ZN, Zodda AR, Yan H, Jackson IL, Polascik TJ, Donatucci CF, Moul JW, Vujaskovic Z, and Koontz BF. Role of oxidative stress in a rat model of radiation-induced erectile dysfunction. J Sex Med 2012;9:1535–1549.     

Cyclin D1 cooperates with p21 to regulate TGFβ-mediated breast cancer cell migration and tumor local invasion

Introduction

Deregulation of the cell cycle machinery is often found in human cancers. Modulations in the cell cycle regulator function and expression result not only in proliferative advantages, but also lead to tumor progression and invasiveness of the cancer. In particular, cyclin D1 and p21 are often over-expressed in human cancers, correlating with high tumor grade, poor prognosis and increased metastasis. This prompted us to investigate the role of the cyclin D1/p21 signaling axis downstream of transforming growth factor beta (TGFβ) in breast cancer progression.

Methods

Cyclins mRNA and protein expressions were assessed by quantitative real-time PCR and Western blot in triple negative breast cancer cell lines. Co-localization and interaction between cyclin D1 and p21 were performed by immunocytochemistry and co-immunoprecipitation, respectively. Cell migration was assessed by wound healing and quantitative time-lapse imaging assays. In addition, the effects of cyclin D1 on cellular structure and actin organization were examined by staining with F-actin marker phalloidin and mesenchymal intermediate filament vimentin. Finally, a mammary fat pad xenograft mouse model was used to assess mammary tumor growth and local invasion.

Results

We found TGFβ to specifically up-regulate the expression of cyclin D1 in triple negative breast cancer cells. Induction of cyclin D1 is also required for TGFβ-mediated cell migration. Suppression of cyclin D1 expression not only resulted in a rounded and epithelial-like phenotype, but also prevented TGFβ-induced vimentin and F-actin co-localization at the cell edge as well as invadopodia formation. Furthermore, TGFβ promoted the nuclear co-localization and physical interaction between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins are required for the initial steps of tumor development, as double knockdown of these two molecules prevented primary tumor formation in a Xenograft mouse model. Moreover, the in vivo studies indicated that locally advanced features of the invasive tumors, including skeletal muscle, mammary fat pad and lymphovascular invasion, as well as ulcerated skin, were attenuated in the absence of cyclin D1 and p21.

Conclusions

Thus, our findings highlight the cyclin D1/p21 signaling axis as a critical regulator of TGFβ-mediated tumor growth initiation and local tumor cell invasion, both in vitro and in vivo.     

Time Course of Recovery of Erectile Function After Radical Retropubic Prostatectomy: Does Anyone Recover After 2 Years?

IntroductionGiven the paucity of literature on the time course of recovery of erectile function (EF) after radical prostatectomy (RP), many publications have led patients and clinicians to believe that erections are unlikely to recover beyond 2 years after RP.AimsWe sought to determine the time course of recovery of EF beyond 2 years after bilateral nerve sparing (BNS) RP and to determine factors predictive of continued improved recovery beyond 2 years.MethodsEF was assessed prospectively on a 5-point scale: (i) full erections; (ii) diminished erections routinely sufficient for intercourse; (iii) partial erections occasionally satisfactory for intercourse; (iv) partial erections unsatisfactory for intercourse; and (v) no erections. From 01/1999 to 01/2007, 136 preoperatively potent (levels 1–2) men who underwent BNS RP without prior treatment and who had not recovered consistently functional erections (levels 1–2) at 24 months had further follow-up regarding EF. Median follow-up after the 2-year visit was 36.0 months.Main Outcome MeasuresRecovery of improved erections at a later date: recovery of EF level 1–2 in those with level 3 EF at 2 years and recovery of EF level 1–3 in those with level 4–5 EF at 2 years.ResultsThe actuarial rates of further improved recovery of EF to level 1–2 in those with level 3 EF at 2 years and to level 1–3 in those with level 4–5 EF at 2 years were 8%, 20%, and 23% at 3, 4, and 5 years postoperatively, and 5%, 17%, and 21% at 3, 4, and 5 years postoperatively, respectively. Younger age was predictive of greater likelihood of recovery beyond 2 years.ConclusionThere is continued improvement in EF beyond 2 years after BNS RP. Discussion of this prolonged time course of recovery may allow patients to have a more realistic expectation. Rabbani F, Schiff J, Piecuch M, Yunis LH, Eastham JA, Scardino PT, and Mulhall JP. Time course of recovery of erectile function after radical retropubic prostatectomy: Does anyone recover after 2 years?.     

Changes in Glitazone Use Among Office-Based Physicians in the U.S., 2003�C2009

OBJECTIVE

Little is known regarding recent changes in glitazone use.

RESEARCH DESIGN AND METHODS

Interrupted time series analyses of nationally representative office-visit data using IMS Health''s National Disease and Therapeutic Index.

RESULTS

From 2003 through 2005, glitazone use increased steadily. From February 2005 to January 2007, rosiglitazone use decreased by 16% (95% CI −20 to −11) annually; pioglitazone use increased at an annual rate of 14% (9–18). During a period of Food and Drug Administration (FDA) advisories, rosiglitazone use declined sharply from 0.42 million monthly treatment visits (February 2007) to 0.13 million monthly visits (May 2008). Pioglitazone use remained stable, accounting for ∼5.8 million physician visits (77% of all glitazone use) where a treatment was used during the final 12 months of observation.

CONCLUSIONS

The combined effect of scientific publications, advisories, and media exposure was associated with a substantial decrease in rosiglitazone use. Despite a class-level FDA advisory, pioglitazone use was not similarly affected.Diabetes treatments change and evolve, and glitazones were rapidly incorporated into practice, with U.S. expenditures reaching $4.2 billion in 2007 (1). Even early evidence suggested rare but serious adverse cardiovascular events with glitazone use (2). Based on accumulating evidence (3), in May 2007 the Food and Drug Administration (FDA) issued an advisory about rosiglitazone''s cardiovascular risks (4,5), followed by a class-wide advisory in August 2007 and an additional rosiglitazone advisory in November 2007 (4).Emerging scientific evidence and the FDA advisories received considerable media coverage, and each of these factors may have been influential in affecting pioglitazone and rosiglitazone use. We examine pioglitazone and rosiglitazone use based on a nationally representative audit of office-based physicians.