DNA Aptamer Raised against Advanced Glycation End Products Improves Sperm Concentration, Motility, and Viability by Suppressing Receptors for Advanced Glycation End Product-Induced Oxidative Stress and Inflammation in the Testes of Diabetic Mice.

Type 2 diabetes mellitus (T2DM) is a risk factor for male infertility, but the underlying molecular mechanisms remain unclear. Advanced glycation end products (AGEs) are pathogenic molecules for diabetic vascular complications. Here, we investigated the effects of the DNA aptamer raised against AGEs (AGE-Apt) on testicular and sperm abnormalities in a T2DM mouse model. KK-Ay (DM) and wild-type (non-DM) 4- and 7-week-old male mice were sacrificed to collect the testes and spermatozoa for immunofluorescence, RT-PCR, and histological analyses. DM and non-DM 7-week-old mice were subcutaneously infused with the AGE-Apt or control-aptamer for 6 weeks and were then sacrificed. Plasma glucose, testicular AGEs, and Rage gene expression in 4-week-old DM mice and plasma glucose, testicular AGEs, oxidative stress, and pro-inflammatory gene expressions in 7-week-old DM mice were higher than those in age-matched non-DM mice, the latter of which was associated with seminiferous tubular dilation. AGE-Apt did not affect glycemic parameters, but it inhibited seminiferous tubular dilation, reduced the number of testicular macrophages and apoptotic cells, and restored the decrease in sperm concentration, motility, and viability of 13-week-old DM mice. Our findings suggest that AGEs-Apt may improve sperm abnormality by suppressing AGE-RAGE-induced oxidative stress and inflammation in the testes of DM mice.

AGE-RAGE Axis Stimulates Oxidized LDL Uptake into Macrophages through Cyclin-Dependent Kinase 5-CD36 Pathway via Oxidative Stress Generation.

Advanced glycation end products (AGEs) are localized in macrophage-derived foam cells within atherosclerotic lesions, which could be associated with the increased risk of atherosclerotic cardiovascular disease under diabetic conditions. Although foam cell formation of macrophages has been shown to be enhanced by AGEs, the underlying molecular mechanism remains unclear. Since cyclin-dependent kinase 5 (Cdk5) is reported to modulate inflammatory responses in macrophages, we investigated whether Cdk5 could be involved in AGE-induced CD36 gene expression and foam cell formation of macrophages. AGEs significantly increased Dil-oxidized low-density lipoprotein (ox-LDL) uptake, and Cdk5 and CD36 gene expression in U937 human macrophages, all of which were inhibited by DNA aptamer raised against RAGE (RAGE-aptamer). Cdk5 and CD36 gene expression levels were correlated with each other. An antioxidant, N-acetyl-l-cysteine, mimicked the effects of RAGE-aptamer on AGE-exposed U937 cells. A selective inhibitor of Cdk5, (R)-DRF053, attenuated the AGE-induced Dil-ox-LDL uptake and CD36 gene expression, whereas anti-CD36 antibody inhibited the Dil-ox-LDL uptake but not Cdk5 gene expression. The present study suggests that AGEs may stimulate ox-LDL uptake into macrophages through the Cdk5-CD36 pathway via RAGE-mediated oxidative stress.

A Dipeptidyl Peptidase-4 Inhibitor Inhibits Foam Cell Formation of Macrophages in Type 1 Diabetes via Suppression of CD36 and ACAT-1 Expression.

Dipeptidyl peptidase-4 (DPP-4) inhibitors have been reported to play a protective role against atherosclerosis in both animal models and patients with type 2 diabetes (T2D). However, since T2D is associated with dyslipidemia, hypertension and insulin resistance, part of which are ameliorated by DPP-4 inhibitors, it remains unclear whether DPP-4 inhibitors could have anti-atherosclerotic properties directly by attenuating the harmful effects of hyperglycemia. Therefore, we examined whether a DPP-4 inhibitor, teneligliptin, could suppress oxidized low-density lipoprotein (ox-LDL) uptake, foam cell formation, CD36 and acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) gene expression of macrophages isolated from streptozotocin-induced type 1 diabetes (T1D) mice and T1D patients as well as advanced glycation end product (AGE)-exposed mouse peritoneal macrophages and THP-1 cells. Foam cell formation, CD36 and ACAT-1 gene expression of macrophages derived from T1D mice or patients increased compared with those from non-diabetic controls, all of which were inhibited by 10 nmol/L teneligliptin. AGEs mimicked the effects of T1D; teneligliptin attenuated all the deleterious effects of AGEs in mouse macrophages and THP-1 cells. Our present findings suggest that teneligliptin may inhibit foam cell formation of macrophages in T1D via suppression of CD36 and ACAT-1 gene expression partly by attenuating the harmful effects of AGEs.

Subcutaneous Infusion of DNA-Aptamer Raised against Advanced Glycation End Products Prevents Loss of Skeletal Muscle Mass and Strength in Accelerated-Aging Mice.

We have developed DNA aptamers that can inhibit the toxic effects of advanced glycation end products (AGE-Apts). We herein evaluated the effects of AGE-Apts on muscle mass and strength in senescence-accelerated mouse prone 8 (SAMP8) mice. Eight-month-old male SAMP8 mice received subcutaneous infusion of control DNA aptamers (CTR-Apts) or AGE-Apts. Mice in an age-matched senescence-accelerated mouse resistant strain 1 (SAMR1) group were treated with CTR-Apts as controls. The soleus muscles were collected after the 8-week intervention for weight measurement and histological, RT-PCR, and immunofluorescence analyses. Grip strength was measured before and after the 8-week intervention. AGE-Apt treatment inhibited the progressive decrease in the grip strength of SAMP8 mice. SAMP8 mice had lower soleus muscle weight and fiber size than SAMR1 mice, which was partly restored by AGE-Apt treatment. Furthermore, AGE-Apt-treated SAMP8 mice had a lower interstitial fibrosis area of the soleus muscle than CTR-Apt-treated SAMP8 mice. The soleus muscle levels of AGEs, oxidative stress, receptor for AGEs, and muscle ring-finger protein-1 were increased in the CTR-Apt-treated mice, all of which, except for AGEs, were inhibited by AGE-Apt treatment. Our present findings suggest that the subcutaneous delivery of AGE-Apts may be a novel therapeutic strategy for aging-related decrease in skeletal muscle mass and strength.

Luseogliflozin inhibits high glucose-induced TGF-ß2 expression in mouse cardiomyocytes by suppressing NHE-1 activity.

OBJECTIVE: Sodium-glucose cotransporter-2 (SGLT2) inhibitors exhibit cardioprotective properties in patients with diabetes. However, SGLT2 is not expressed in the heart, and the underlying molecular mechanisms are not fully understood. We investigated whether the SGLT2 inhibitor luseogliflozin exerts beneficial effects on high glucose-exposed cardiomyocytes via the suppression of sodium-hydrogen exchanger-1 (NHE-1) activity. METHODS: Mouse cardiomyocytes were incubated under normal or high glucose conditions with vehicle, luseogliflozin, or the NHE-1 inhibitor cariporide. NHE-1 activity and gene expression were evaluated by the SNARF assay and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, respectively. Six-week-old male db/db mice were treated with vehicle or luseogliflozin for 6 weeks, and the hearts were collected for histological, RT-PCR, and western blot analyses. RESULTS: High glucose increased NHE-1 activity and transforming growth factor (Tgf)-ß2 mRNA levels in cardiomyocytes, both of which were inhibited by luseogliflozin or cariporide, whereas their combination showed no additive suppression of Tgf-ß2 mRNA levels. Luseogliflozin attenuated cardiac hypertrophy and fibrosis in db/db mice in association with decreased mRNA and protein levels of TGF-ß2. CONCLUSIONS: Luseogliflozin may suppress cardiac hypertrophy in diabetes by reducing Tgf-ß2 expression in cardiomyocytes via the suppression of NHE-1 activity.

Glucose-Dependent Insulinotropic Polypeptide Suppresses Foam Cell Formation of Macrophages through Inhibition of the Cyclin-Dependent Kinase 5-CD36 Pathway.

Glucose-dependent insulinotropic polypeptide (GIP) has been reported to have an atheroprotective property in animal models. However, the effect of GIP on macrophage foam cell formation, a crucial step of atherosclerosis, remains largely unknown. We investigated the effects of GIP on foam cell formation of, and CD36 expression in, macrophages extracted from GIP receptor-deficient (Gipr-/-) and Gipr+/+ mice and cultured human U937 macrophages by using an agonist for GIP receptor, [D-Ala2]GIP(1-42). Foam cell formation evaluated by esterification of free cholesterol to cholesteryl ester and CD36 gene expression in macrophages isolated from Gipr+/+ mice infused subcutaneously with [D-Ala2]GIP(1-42) were significantly suppressed compared with vehicle-treated mice, while these beneficial effects were not observed in macrophages isolated from Gipr-/- mice infused with [D-Ala2]GIP(1-42). When macrophages were isolated from Gipr+/+ and Gipr-/- mice, and then exposed to [D-Ala2]GIP(1-42), similar results were obtained. [D-Ala2]GIP(1-42) attenuated ox-LDL uptake of, and CD36 gene expression in, human U937 macrophages as well. Gene expression level of cyclin-dependent kinase 5 (Cdk5) was also suppressed by [D-Ala2]GIP(1-42) in U937 cells, which was corelated with that of CD36. A selective inhibitor of Cdk5, (R)-DRF053 mimicked the effects of [D-Ala2]GIP(1-42) in U937 cells. The present study suggests that GIP could inhibit foam cell formation of macrophages by suppressing the Cdk5-CD36 pathway via GIP receptor.

Glucose-dependent insulinotropic polypeptide inhibits cardiac hypertrophy and fibrosis in diabetic mice via suppression of TGF-ß2.

Diabetic cardiomyopathy is associated with an increased risk for heart failure and death in patients with diabetes. We investigated here whether and how GIP attenuated cardiac hypertrophy and fibrosis in diabetic mice with obesity. Diabetic db/db mice at 7 weeks old were infused with vehicle or GIP (50 nmol/kg/day) for 6 weeks, and hearts were collected for histological and RT-PCR analyzes. Cardiomyocytes isolated from neonatal mice were incubated with or without 300 nM [D-Ala2]-GIP, 30 mM glucose, or 100 µg/mL advanced glycation end products (AGEs) for RT-PCR and lucigenin assays. Compared with non-diabetic mice, diabetic mice exhibited larger left ventricle wall thickness and cardiomyocyte sizes and more fibrotic areas in association with up-regulation of myosin heavy chain ß (ß-Mhc) and transforming growth factor-beta2 (Tgf-ß2) mRNA levels, all of which were inhibited by GIP infusion. High glucose increased NADPH oxidase-driven superoxide generation and up-regulated ß-Mhc, Tgf-ß2, and receptor for AGEs mRNA levels in cardiomyocytes, and augmented the AGE-induced ß-Mhc gene expression. [D-Ala2]-GIP attenuated all of the deleterious effects of high glucose and/or AGEs on cardiomyocytes. Our present findings suggest that GIP could inhibit cardiac hypertrophy and fibrosis in diabetic mice via suppression of TGF-ß2.

Glucose Variability is Independently Correlated with Serum Level of Pigment Epithelium-Derived Factor in Type 2 Diabetes.

INTRODUCTION: Pigment epithelium-derived factor (PEDF) may play a role in cardiometabolic disorders. The aim of this study was to investigate which biochemical and clinical parameters are independently associated with serum PEDF levels in patients with type 2 diabetes mellitus (T2DM). METHODS: We performed a cross-sectional analysis of 124 patients with T2DM who underwent continuous glucose monitoring (CGM) and blood chemistry analysis, including the diacron-reactive oxygen metabolites (d-ROMs) test and serum PEDF measurement (study 1). Then we investigated whether the changes in the studied biochemical and clinical parameters after 24 weeks of treatment (Δparameters) with anti-hyperglycemic agents, including sodium-glucose cotransporter 2 inhibitors, glucagon-like peptide 1 receptor agonists, and/or insulin and anti-hypertensive drugs and statins, were independently correlated with change in PEDF (ΔPEDF) in 52 of the patients with T2DM for whom there was sufficient serum samples to perform the post-treatment analysis (study 2). Serum levels of PEDF were measured with an enzyme-linked immunosorbent assay. CGM metrics were calculated on days 2 and 3. Oxidative stress was evaluated using the d-ROMs test. RESULTS: Body mass index (BMI), triglycerides, fasting C-peptide, mean amplitude of glycemic excursions (MAGE), urinary albumin-to-creatinine ratio (UACR), and d-ROMs were positively associated with serum PEDF level, and high-density lipoprotein cholesterol (HDL-C) and estimated glomerular filtration rate (eGFR) were inversely associated with serum PEDF level. Because these parameters were correlated with each other, multivariate stepwise analysis was performed: eGFR, HDL-C, BMI, MAGE, and UACR remained significant (R2 = 0.452). Furthermore, ΔMAGE and Δd-ROMs were positively correlated with ΔPEDF in study 2. CONCLUSIONS: The results of this study suggest that MAGE may be independently correlated with elevations in serum PEDF level in patients with T2DM.

Anti-inflammatory and atheroprotective properties of glucagon.

Although glucagon has been shown to exert pleiotropic actions in various types of cells and organs through the interaction with its receptor, its pathophysiological role in atherosclerotic cardiovascular disease remains unclear. Here, we examined whether and how glucagon could attenuate the progression of atherosclerotic plaques in apolipoprotein E-deficient mice (ApoE-/-), an animal model of atherosclerosis. Glucagon (138 or 413 nmol/kg/day) or vehicle was infused to mice at 16 weeks of age. After 4-week treatment, vascular samples were collected for histological and RT-PCR analyses. Human monocytic THP-1 cells were pre-incubated with or without a glucagon receptor antagonist L-168049, and then treated with or without glucagon for 7 h. Gene and protein expressions were determined by RT-PCR and western blot analyses, respectively. High-dose glucagon infusion significantly decreased aortic plaque area and volume in ApoE-/- mice, both of which were inversely correlated with plasma glucagon levels. Glucagon infusion also reduced the ratio of pro-inflammatory interleukin-1ß to anti-inflammatory interleukin-10 gene expression in aortae. Glucagon receptor was expressed in THP-1 cells, and 1 nM glucagon decreased the ratio of interleukin-1ß to interleukin-10 gene expression, which was significantly prevented by L-168049. Our present findings suggest that glucagon could exert atheroprotection partly via its anti-inflammatory property.

Emphysematous cystitis in an elderly Japanese patient with type 2 diabetes mellitus.

We report a rare but fatal case of emphysematous cystitis (EC) that presented as a typical image of gas bubbles within the urinary bladder wall on computed tomography (CT). This disease has a high mortality rate more than that reported previously based on recent literature review in Japan.

A Dipeptidyl Peptidase-4 Inhibitor Suppresses Macrophage Foam Cell Formation in Diabetic db/db Mice and Type 2 Diabetes Patients.

Dipeptidyl peptidase-4 (DPP-4) inhibitors could have antiatherosclerotic action, in addition to antihyperglycemic roles. Because macrophage foam cells are key components of atherosclerosis, we investigated the effect of the DPP-4 inhibitor teneligliptin on foam cell formation and its related gene expression levels in macrophages extracted from diabetic db/db (C57BLKS/J Iar -+Leprdb/+Leprdb ) mice and type 2 diabetes (T2D) patients ex vivo. We incubated mouse peritoneal macrophages and human monocyte-derived macrophages differentiated by 7-day culture with oxidized low-density lipoprotein in the presence/absence of teneligliptin (10 nmol/L) for 18 hours. We observed remarkable suppression of foam cell formation by teneligliptin treatment ex vivo in macrophages isolated from diabetic db/db mice (32%) and T2D patients (38%); this effect was accompanied by a reduction of CD36 (db/db mice, 43%; T2D patients, 46%) and acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) gene expression levels (db/db mice, 47%; T2D patients, 45%). Molecular mechanisms underlying this effect are associated with downregulation of CD36 and ACAT-1 by teneligliptin. The suppressive effect of a DPP-4 inhibitor on foam cell formation in T2D is conserved across species and is worth studying to elucidate its potential as an intervention for antiatherogenesis in T2D patients.