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AimsTo establish the sequential changes by glycemic control in the mean thickness, volume and reflectance of the macular photoreceptor layers (MPRLs) and retinal pigment epithelium in patients with type 2 diabetes without diabetic retinopathy.MethodsThirty-one poorly controlled (HbA1c > 8.0%) patients with type 2 diabetes without diabetic retinopathy undergoing glycemic control and 39 control subjects with normal HbA1c levels (< 5.9%) underwent periodical full medical, neurological and ophthalmological examinations over 2 years. Glycemic variability was evaluated by standard deviation and coefficient of variation of monthly measured HbA1c levels and casual plasma glucose. 3D swept source-optical coherence tomography (OCT) and OCT-Explorer-generated enface thickness, volume and reflectance images for 9 subfields defined by Early Treatment Diabetic Retinopathy Study of 4 MPRLs {outer nuclear layer, ellipsoid zone, photoreceptor outer segment (PROS) and interdigitation zone} and retinal pigment epithelium were acquired every 3 months.ResultsGlycemic control sequentially restored the thickness and volume at 6, 4 and 5 subfields of outer nuclear layer, ellipsoid zone and PROS, respectively. The thickness and volume of outer nuclear layer were restored related to the decrease in HbA1c and casual plasma glucose levels, but not related to glycemic variability and neurological tests. The reflectance of MPRLs and retinal pigment epithelium in patients was marginally weaker than controls, and further decreased at 6 or 15 months during glycemic control. The reduction at 6 months coincided with high HbA1c levels.ConclusionGlycemic control sequentially restored the some MPRL thickness, especially of outer nuclear layer. In contrast, high glucose during glycemic control decreased reflectance and may lead to the development of diabetic retinopathy induced by glycemic control. The repeated OCT examinations can clarify the benefit and hazard of glycemic control to the diabetic retinopathy.

The incidence of both type 1 and type 2 diabetes is increasing worldwide. Diabetic peripheral neuropathy (DPN) is among the most distressing and costly of all the chronic complications of diabetes and is a cause of significant disability and poor quality of life. This incurs a significant burden on health care costs and society, especially as these young people enter their peak working and earning capacity at the time when diabetes-related complications most often first occur. DPN is often asymptomatic during the early stages; however, once symptoms and overt deficits have developed, it cannot be reversed. Therefore, early diagnosis and timely intervention are essential to prevent the development and progression of diabetic neuropathy. The diagnosis of DPN, the determination of the global prevalence, and incidence rates of DPN remain challenging. The opinions vary about the effectiveness of the expansion of screenings to enable early diagnosis and treatment initiation before disease onset and progression. Although research has evolved over the years, DPN still represents an enormous burden for clinicians and health systems worldwide due to its difficult diagnosis, high costs related to treatment, and the multidisciplinary approach required for effective management. Therefore, there is an unmet need for reliable surrogate biomarkers to monitor the onset and progression of early neuropathic changes in DPN and facilitate drug discovery. In this review paper, the aim was to assess the currently available tests for DPN’s sensitivity and performance.

Aims: To investigate the impact of thinning at individual grids of macular neuroretinal layers, clinical factors, and inadequate light exposure on the specific components of sleep disorder in patients with type 2 diabetes. Methods: One hundred twenty-four patients with type 2 diabetes without clinical evidences of diabetic retinopathy and neuropathy (HbA1c: 8.3%, diabetes duration; 8.7 years) and 54 age- and sex-matched control subjects (HbA1c: 5.6%) underwent detailed clinical, neurological, and ophthalmological examinations. The sleep disorder was assessed by the Pittsburgh Sleep Quality Index Japanese Version (PSQI-J). The temporal structures of daily life were assessed by the Munich Chronotype Questionnaire Japanese Version. The thickness at nine grids defined by the Early Treatment Diabetic Retinopathy Study of nine macular neuroretinal layers was determined by swept-source optical coherence tomography and OCT-Explorer. The associations between the individual components of sleep disorders and the thickness at each grid of macular neuroretinal layers, clinical factors, or the temporal structures of daily life were examined. Results: The prevalence of the sleep disorder, global score, and four individual PSQI-J scores in patients with type 2 diabetes were higher than control subjects. The thickness of two and five grids of two inner retinal layers and four to seven grids of four outer retinal layers in patients with type 2 diabetes was thinner than those in control subjects. The thickness at one to eight grids of four outer retinal layers in type 2 diabetic patients was inversely associated with global score and five individual scores of sleep disorder. The thinning at one to two grids of the inner plexiform layer was related to three high individual scores of sleep disorder. The inappropriate light exposure was associated with the sleep disorder and altered macular neuroretinal layers. The high HbA1c and LDL-cholesterol levels were related to the high global score and two individual scores of sleep disorder, respectively. Conclusion: In patients with type 2 diabetes, the thinning at grids of the inner plexiform layer and outer retinal layers was associated with the high scores of specific components of the sleep disorder. The sleep disorder was also related to hyperglycemia, dyslipidemia, and inappropriate light exposure.

OBJECTIVE To investigate the impact of normalizing HbA1c by extensive HbA1c control (EHC) on neuropathy outcome measures (NOMs), nephropathy, and retinopathy in type 2 diabetes. RESEARCH DESIGN AND METHODS Detailed clinical and neurological examinations were performed in two cohorts of 38 patients with uncontrolled type 2 diabetes (HbA1c 9.6% [81.4 mmol/mol]) at baseline and after glycemic control (GC) with or without EHC by diet restriction and hypoglycemic agents over 4 years along with 48 control subjects with normal glucose tolerance (NGT) and 34 subjects with impaired glucose tolerance (IGT) only at baseline. EHC patients, control subjects, and subjects with IGT underwent oral glucose tolerance tests. Glycemic variability (GV) was evaluated by SD and coefficient of variation of monthly measured HbA1c levels and casual plasma glucose. RESULTS In the EHC cohort, HbA1c levels over 4.3 years and the last 2 years improved to 6.1% (43.2 mmol/mol) and 5.8% (39.9 mmol/mol) with 7.3 kg body wt reduction, and 50% and 28.9% of patients returned to IGT and NGT, respectively, at end point. Baseline neurophysiological and corneal nerve fiber (CNF) measures were impaired in patients. Normalized HbA1c with EHC improved neurophysiological and CNF measures to be similar for those for IGT, while GC without EHC (mean HbA1c level 7.0% [53.5 mmol/mol]) improved only vibration perception. The mean normalized HbA1c levels by EHC determined NOM improvements. The high GV and baseline HbA1c levels compromised NOMs. Albumin excretion rate significantly decreased, while retinopathy severity and frequency insignificantly worsened on EHC. CONCLUSIONS Normalizing HbA1c in type 2 diabetes of short duration improves microvascular complications including neuropathy and nephropathy more effectively than standard GC but not retinopathy.

The aim of this study was to investigate the impact of extensive HbA1c control on major microvascular complications including neuropathy, retinopathy and nephropathy in T2DM patients with short duration. Detailed Clinical (e.g., full Lipid profile test, BMI, Blood Pressure, eGFR), ophthalmic (e.g., Fundus Photography, Optical Coherence Topography (OCT), Corneal Confocal Microscopy (CCM)) and neurological examinations (e.g., symptoms, electrophysiology, quantitative sensory testing, Cardiac Autonomic Neuropathy tests) were performed in 76 patients with T2DM at baseline and after glycemic control (GC) with or without Extensive HbA1C control (EHC) over 4 years along with 48 controls with normal glucose tolerance (NGT) and 34 subjects with impaired glucose tolerance (IGT). The mean HbA1c levels during whole follow-up period were closely related with the improvement in neurological markers. The multiple regression analysis indicates that the changes in all Corneal Nerve markers (CNF) and neurophysiological measures were closely associated with the mean HbA1c level over whole follow-up period. Higher SD and CV of HbA1c levels were negatively associated with some CNF and neurophysiological measures. The high baseline HbA1c levels deteriorated many neurological markers. In this study patients with poorly controlled type 2 diabetes returned to IGT by normalizing HbA1c level for 2 years without hypoglycemia. Nephropathy reduced from 34.2% to 18.4%. However, the prevalence of retinopathy progressed from 7.9% at baseline to 13.2%. Prevalence of neuropathy reduced from 13.2% to 2.6%. Although improvement of neurological markers in this study was significant, but the changes were small. The intensive GC in T2DM with short duration is associated with a reduction in microvascular complications (mostly albuminuria). The normalized HbA1c levels are more effective than standard care for preventing the development of neuropathy, but not retinopathy. Disclosure M. Tavakoli: None. F. Ishibashi: None.

This study aims to establish the corneal nerve fiber (CNF) morphological alterations in a large cohort of type 2 diabetic patients and to investigate the association between the bead size, a novel parameter representing composite of accumulated mitochondria, glycogen particles, and vesicles in CNF, and the neurophysiological dysfunctions of the peripheral nerves. 162 type 2 diabetic patients and 45 healthy control subjects were studied in detail with a battery of clinical and neurological examinations and corneal confocal microscopy. Compared with controls, patients had abnormal CNF parameters. In particular the patients had reduced density and length of CNF and beading frequency and increased bead size. Alterations in CNF parameters were significant even in patients without neuropathy. The HbA1c levels were tightly associated with the bead size, which was inversely related to the motor and sensory nerve conduction velocity (NCV) and to the distal latency period of the median nerve positively. The CNF density and length positively correlated with the NCV and amplitude. The hyperglycemia-induced expansion of beads in CNF might be a predictor of slow NCV in peripheral nerves in type 2 diabetic patients.

To investigate the impact of normalizing HbADetailed clinical and neurological examinations were performed in two cohorts of 38 patients with uncontrolled type 2 diabetes (HbAIn the EHC cohort, HbANormalizing HbA

Aim: Hyperglycemia is associated with increased risk of microvascular complications in patients with type 2 diabetes (T2DM). The aim of the present study is to investigate whether reduction of the levels of HbA1c by tight glycemic control (GC) decreases the rate of microvascular complications and improves the neurological measures in T2DM. Methods: Detailed clinical, neurological examinations including corneal confocal microscopy (CCM), were performed in 141 patients with poorly controlled type 2 diabetes (mean age 53, Duration diabetes: 9 years) at the baseline and 60 age-matched control subjects and followed-up with GC for 4 years. Nephropathy and Retinopathy also assessed. Patients were stratified according to mean HbA1c level during follow-up. Results: At baseline CCM showed significant nerve fiber damage in all patients compared to controls. Despite strict GC and improvement in mean HbA1c by around 2.8%, overall the cumulative incidence of neuropathy increased from 17.7% at baseline to 21.3% and retinopathy increased from 21.3% to 35.5%, however the cumulative incidence of nephropathy reduced from 37.6% to 22%. Tight GC was effective in improving Neuropathy only in those with very poor Control at the Baseline. The interval changes in HbA1c levels were correlated with the interval changes in CNF measures and neurophysiological tests. The results showed one percent reduction in HbA1c levels per year significantly improved CNF measures and neurophysiological dysfunctions. Based on the results of this study, for improving neuropathy, only HbA1c close to 6.5% is effective for improvement in structure and function of the corneal nerves. Conclusion: This study showed that the GC was just improved nephropathy among microvascular complications. Glucose control did only improve neurophysiological and corneal nerve measurements when near-normoglycemia was reached. Despite tight GC, the retinopathy progressed in patients with type 2 diabetes at this cohort. Disclosure M. Tavakoli: None. F. Ishibashi: None.

Aims Hyperglycemia is associated with an increased risk of microvascular complications in patients with type 2 diabetes. The aim of the present study was to investigate whether the reduction of the levels of HbA1c by tight glycemic control (GC) decreases the rate of microvascular complications and improves the neurological measures in patients with type 2 diabetes. Methods Detailed clinical and neurological examinations including corneal confocal microscopy (CCM) were performed in 141 Japanese patients with type 2 diabetes and 60 age-matched control subjects at baseline and follow-up with GC for 4 years. Patients were stratified according to the mean HbA1c level during follow-up into good (HbA1c

Aims/Introduction To measure the elasticity of the tibial nerve using sonoelastography, and to associate it with diabetic neuropathy severity, the cross-sectional area of the tibial nerve and neurophysiological findings in type 2 diabetic patients. Materials and Methods The elasticity of the tibial nerve was measured as the tibial nerve:acoustic coupler strain ratio using high-resolution ultrasonography in 198 type 2 diabetic patients stratified into subgroups by neuropathy severity, and 29 control participants whose age and sex did not differ from the diabetic subgroups. Results The elasticity of the tibial nerve in patients without neuropathy (P

The main aim of the present paper is to examine whether the pupillary light reflex (PLR) mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs) is impaired in type 2 diabetic patients. One hundred and three diabetic patients without diabetic autonomic neuropathy (DAN) and 42 age-matched controls underwent a series of detailed neurological examinations. The patients were stratified into three groups: stage I, no neuropathy; stage II, asymptomatic neuropathy; stage III, symptomatic but without DAN. The PLR to 470 and 635 nm light at 20 cd/m2was recorded. Small fiber neuropathy was assessed by corneal confocal microscopy and quantifying corneal nerve fiber (CNF) morphology. The 470 nm light induced a stronger and faster PLR than did 635 nm light in all subjects. The PLR to both lights was impaired equally across all of the diabetic subgroups. The postillumination pupil response (PIPR) after 470 nm light offset at ≥1.7 sec was attenuated in diabetic patients without differences between subgroups. Receiver operating characteristic analysis revealed that the PIPR mediated by ipRGCs in patients with stage II and stage III neuropathy was different from that of the control subjects. Clinical factors, nerve conduction velocity, and CNF measures were significantly correlated with PLR parameters with 470 nm light. PLR kinetics were more impaired by stimulation with blue light than with red light in diabetic patients without DAN.

Oxidative stress, which is provoked in patients with diabetes, plays critical roles in the pathogenesis of coronary heart disease (CHD). We simultaneously determined 5 relatively common genetic variants related to oxidative stress and evaluated the combined effect on CHD.We enrolled 1977 Japanese type 2 diabetic subjects without history of CVD (males 66.1%, 59.5 ± 10.0 years old), determined their genotypes regarding glutamate-cysteine ligase modifier subunit (GCLM) C-588T, manganese superoxide dismutase (SOD2) Val16Ala, endothelial nitric oxide synthase (NOS3) G894T, NAD(P)H oxidase p22phox (CYBA) C242T, and myeloperoxidase (MPO) G-463A polymorphisms, and prospectively evaluated the association between these polymorphisms and CHD events.The median follow-up period was 7.5 years and there were 85 new CHD events. The single association analysis revealed that there were no statistically significant associations between each polymorphism and the prevalence of CHD. Interestingly, the risk of CHD event was higher with the increase of the total number of 10 concomitant unfavorable "pro-oxidant alleles" in each subject (p for trend = 0.018, log-rank test). Especially, the carriers of ≥8 pro-oxidant alleles had a significantly increased risk as compared to the carriers of8 pro-oxidant alleles, whether the other clinical variables were adjusted (HR 2.92 with 95%CI 1.50-5.67, p = 0.002) or not (HR 2.89 with 95%CI 1.49-5.59, p = 0.002)..Accumulation of gene polymorphisms related to oxidative stress is likely associated with the development of CHD in patients with type 2 diabetes, suggesting that the combined information about these variants is useful to assess the risk of CHD.

Aims/Introduction To study the correlation between sudomotor function, sweat gland duct size and corneal nerve fiber pathology in type 2 diabetes. Materials and Methods Sudomotor function was quantified by Neuropad test, and sweat gland duct and corneal nerve fibers were visualized by confocal microscopy in 78 patients with type 2 diabetes stratified by diabetic neuropathy and 28 control participants. Results In patients with diabetic neuropathy, sudomotor function, as judged by the time required for complete color change of a Neuropad, was impaired compared with that of controls (P

Aims/Introduction We compared the morphometric features of corneal epithelial basal cells between patients with type 2 diabetes mellitus and healthy controls, and analyzed the relationship of these features with corneal nerve fiber pathology and clinical factors in the patients. Materials and Methods Corneal epithelial basal cells and corneal nerve fibers were visualized by corneal confocal microscopy in 75 patients with type 2 diabetes and 42 age-matched controls. Density, area and area variability of corneal epithelial basal cells, as well as the width of the intercellular space between neighboring cells, were evaluated for both groups. Results Patients showed decreased density (P

Objective Adiponectin has anti-atherogenic properties and reduced serum adiponectin levels are associated with cardiovascular disease (CVD). In this study, we examined the relationship between CVD and adiponectin ( ADIPOQ ) gene G276T polymorphism that is associated with serum adiponectin level in a large cohort of type 2 diabetic patients. Research design and methods We enrolled 2637 Japanese type 2 diabetic subjects (males, 61.1%; age, 54.9±7.9 years old), determined their genotypes regarding ADIPOQ G276T polymorphisms, and evaluated the association between this polymorphism and the prevalence of CVD (myocardial infarction and/or cerebral infarction). Results The prevalence of CVD tended to be higher as the number of G alleles increased [GG (9.5%), GT (6.8%), TT (5.6%), p value for trend=0.0059] and was significantly higher in the subjects with GG genotype compared to those with GT or TT genotype (9.5% vs. 6.6%, p =0.0060). Multiple logistic regression analyses revealed that the number of G alleles (Odds ratio (OR)=1.49 with 95%CI 1.09–2.05, p =0.0125) and GG genotype (OR=1.66 with 95%CI 1.13–2.43, p =0.0098) were significantly associated with CVD even after adjustment for conventional risk factors. Interestingly, the presence of obesity further and significantly increased the risk of CVD in the subjects with GG genotype (OR=1.67 with 95%CI 1.14–2.44, p =0.0090) but not in the subjects with TT or GT genotype (OR=1.17 with 95%CI 0.73–1.89, NS). Conclusions It is likely that the G allele of the ADIPOQ G276T polymorphism is a susceptibility allele for CVD in Japanese type 2 diabetic patients, especially when they accompany obesity.

It is likely that the C allele of the polymorphism at position 29 of the translated sequence of transforming growth factor (TGF)-β1 gene, which codes a pleiotropic cytokine expressed in a variety of cells, is a susceptibility allele for cerebral infarction in Japanese type 2 diabetic patients.

Aims/Introduction: Morphological changes to corneal C-fibers in Japanese type 1 diabetic patients were visualized by corneal confocal microscopy (CCM). The effects of prior glycemic control and blood pressure on morphological parameters were clarified. Materials and Methods: Corneal nerve fibers were visualized by CCM in 38 Japanese type 1 diabetic patients (14 with and 24 without neuropathy) and 38 controls. Morphological parameters were compared and related to annual mean HbA1c, blood pressure, and serum lipid levels of previous years prior to CCM examination. Results: Compared with controls, diabetic patients had reduced corneal nerve fiber length (CNFL; 9.80 ± 0.38 vs 13.65 ± 0.88 mm/mm2; P

It is likely that the C allele of the polymorphism at position −106 in the promoter of aldose reductase gene, which codes a rate-limiting enzyme of the polyol pathway, is a susceptibility allele for diabetic retinopathy in Japanese type 2 diabetic patients.

We examined the association between diabetic retinopathy and monocyte chemoattractant protein (MCP)-1 A-2518G polymorphism in 3802 Japanese type 2 diabetic subjects. The prevalence of diabetic retinopathy was higher as the number of G alleles increased, suggesting that the G allele of this polymorphism is a susceptibility allele for diabetic retinopathy.

OBJECTIVE It is believed that disruption of vulnerable atherosclerotic plaque and subsequent thrombus formation play critical roles in the pathogenesis of cerebral infarction. We simultaneously determined four relatively common genetic variants related to plaque rupture or subsequent local thrombus formation and evaluated the combined effect on cerebral infarction. RESEARCH DESIGN AND METHODS We enrolled 3,094 Japanese type 2 diabetic subjects (62.7% male; aged 61.5 ± 8.4 years) and determined their genotypes regarding matrix metalloproteinase 9 C-1562T, coagulation factor XII (F12) C46T, von Willebrand factor (VWF) G-1051A, and plasminogen activator inhibitor (PAI-1) 675 4G/5G polymorphisms. The diagnosis of cerebral infarction was performed based on history, physical examination, and neuroimaging. RESULTS The single association analysis revealed that there were no statistically significant associations between each polymorphism and the prevalence of cerebral infarction. Interestingly, the prevalence of cerebral infarction was higher with the increase of the total number of four concomitant unfavorable proatherothrombotic alleles in each subject (P value for linear trend = 0.004). Furthermore, a multiple logistic regression analysis showed that the number of proatherothrombotic alleles was a risk factor for cerebral infarction independently of conventional risk factors (odds ratio for one-point increase in the number of proatherothrombotic allele 1.15 [95% CI 1.05–1.26], P = 0.004). CONCLUSIONS Accumulation of gene polymorphisms related to plaque rupture and thrombus formation is likely associated with the prevalence of cerebral infarction in type 2 diabetic patients, suggesting that the combined information about these variants is useful to assess the risk of cerebral infarction.

It is well known that oxidative stress plays critical roles in the pathogenesis of atherosclerosis. In this study, we enrolled 1746 type 2 diabetic subjects, determined 4 common genetic variants related to oxidative stress (glutamate-cysteine ligase modifier subunit (GCLM) C-588T, myeloperoxidase G-463A, human paraoxonase 1 Gln192Arg and NAD(P)H oxidase p22phox C242T polymorphisms), and measured carotid intima-media thickness (IMT) as a surrogate marker for atherosclerosis. GCLM C-588T polymorphism was associated with average IMT (AveIMT) (r=0.090, p=0.0008), but the association between the other 3 polymorphisms and AveIMT did not reach the statistical significance. However, AveIMT was significantly greater as the total number of 4 concomitant "pro-oxidant alleles" in each subject was increased (r=0.108, p0.0001). Furthermore, the number of "pro-oxidant alleles" was a risk factor for a high AveIMT independently of conventional risk factors (p=0.0003). In conclusion, accumulation of oxidative stress-associated alleles was associated with carotid atherosclerosis in type 2 diabetic patients.

In proximal tubular epithelial cells (PTECs), depolymerization of actin by cofilin plays a crucial role in maintaining polarity and function. Cofilin is inactivated when phosphorylated by p-Lin-11/Isl-1/Mec-3 kinase (LIMK) to give p-cofilin. LIMK is phosphorylated by phosphorylated p21-activated kinase (PAK), a downstream signal of phosphoinositide 3-kinase (PI3K), or by Rho kinase (ROCK), and is dephosphorylated by slingshot (SSH). However, in PTECs the signaling pathways regulating phosphorylation and dephosphorylation of cofilin, and the influence of high glucose (HG) on these pathways remain to be elucidated. Here, we show that HG in cultured porcine PTECs (LLC-PK1) increases p-cofilin and p-LIMK1 beyond 6 h and that the simultaneous presence of phlorizin reverses the increase. HG did not influence the levels of PI3K-p85, downstream signals to SSH1 and p-PAK1, and mRNA of cofilin, LIMK1 and SSH1. On the other hand, wortmannin and LY294002 markedly increased p-cofilin and p-LIMK1 without influencing on the level of SSH1 protein. HG-activated RhoA and ROCK2 beyond 3 h, and phlorizin attenuated this activation. GF109203X inhibited HG-induced increase in membranous RhoA and ROCK2, and phorbol ester increased these proteins. Y27632 (a ROCK inhibitor) reversed HG-induced increases of p-cofilin and p-LIMK1. We conclude that HG increases p-cofilin by phosphorylating LIMK1 through activation of Rho/Rho kinase, probably due to diacylglycerol-sensitive PKC activation resulting from increased glucose influx. HG did not alter PI3K or its downstream signals, even though PI3K has a physiological role in maintaining the cofilin level by activating SSH1.

Aims/Introduction To evaluate the morphological changes of the median and posterior tibial nerve using high-resolution ultrasonography, and the corneal C fiber pathology by corneal confocal microscopy in type 2 diabetic patients. Materials and Methods The cross-sectional area, hypoechoic area and maximum thickness of the nerve fascicle of both nerves were measured by high-resolution ultrasonography in 200 type 2 diabetic patients, stratified by the severity of diabetic neuropathy, and in 40 age- and sex-matched controls. These parameters were associated with corneal C fiber pathology visualized by corneal confocal microscopy, neurophysiological tests and severity of diabetic neuropathy. Results The cross-sectional area, hypoechoic area and maximum thickness of the nerve fascicle of both nerves in patients without diabetic neuropathy were larger than those in control subjects (P

Purpose. Transforming growth factor -s2 (TGF-s2) is a predominant isoform of TGF-ss in the eye and plasmin is a peptidase with many functions. To better understand the pathogenesis of retinal microcirculation disorders, the effects of TGF-s2 and plasmin on cultured bovine retinal pericytes were investigated. Methods. Exogenous TGF-s2 or plasmin was added to some cultures, DNA synthesis during cell cycle progression was investigated using [ 3 H]thymidine incorporation. Anti-TGF-s2 antibody was added to neutralize the effects of TGF-s2. TGF-s2 in the culture medium was measured using enzyme-linked immunosorbent assay (ELISA). Results. Exogenous TGF-s2 (10 pg to 100 ng/mL) suppressed DNA synthesis. Pericytes produced TGF-s2. Anti-TGF-s2 antibody neutralized TGF-s2 and accelerated DNA synthesis, which shows that pericytes regulate their own cell cycle by action of the autocrine and/or paracrine system of TGF-s2. Plasmin (0.2 to 0.5 U/mL) accelerated DNA synthesis in a dose-dependent manner, while addition of ...

After an intravenous infusion Of L-arginine to inhibit tubular reabsorption of albumin, glomerular clearance, renal clearance, and tubular reabsorption of unmodified albumin (UMA) and glycated albumin (GA) were determined in 72 patients with NIDDM without (NIDDM-I; n = 47) or with microalbuminuria (NIDDM-II; n = 25) and in 24 healthy control subjects. Samples of serum albumin and dialyzed urine obtained 60 min before and during L-arginine infusion were applied to an affinity column to separate GA from UMA, and their albumin contents were assayed. The serum level of GA in NIDDM patients was higher than that in control subjects (P0.0001). Both UMA and GA were excreted in excess in NIDDM-II as compared with the other two groups (P0.0001), and UMA comprised 80% of total albumin excretion. In NIDDM-II, the glomerular clearance of UMA (2.5 +/- 0.16NIDDM-I [1.8 +/- 0.1]control subjects [1.3 +/- 0.1 microliter/min], P0.001) and of GA (1.7 +/- 0.13NIDDM-I = control subjects [1.1 +/- 0.1 microliter/min], P0.001) were enhanced, as compared with the other two groups. Renal clearance of UMA (1.3 +/- 0.13 microliter/min) and GA (0.89 +/- 0.09 microliter/min) in NIDDM-II was greater than that in control subjects (0.27 +/- 0.03, 0.19 +/- 0.02 microliter/min) or in NIDDM-I (0.30 +/- 0.03, 0.11 +/- 0.01 microliter/min). Tubular reabsorption of UMA, as assessed by the difference between glomerular and renal clearances of albumin, in NIDDM-II (1.1 +/- 0.1 microliter/min) was less than in NIDDM-I (1.50 +/- 0.09 microliter/min), and that of GA in NIDDM-II was lower than that in the other two groups, despite exaggerated glomerular clearance of GA and UMA in NIDDM-II. In summary, microalbuminuria in NIDDM is caused by increased excretion of UMA resulting from the decompensated ability of tubular reabsorption, which is exceeded by increased glomerular clearance of UMA.

To determine whether the offspring of hypertensive patients (OHP) might have a higher serum insulin level than offspring of normotensive parents, we studied 152 girls in junior high school, 21 of whom had at least one hypertensive parent. The remaining 131 girls had normotensive parents and served as the controls. After an overnight fast, we measured the subjects' height, body weight, blood pressure and body fat mass, and collected blood for assay of serum immunoreactive insulin (IRI), lipids and apolipoproteins. Despite a similar body mass index, the OHP had a significantly greater body fat mass (P < 0.05) and % fat mass (P < 0.05) than the controls. The OHP exhibited a significantly higher serum IRI level than the controls (P < 0.05), but no differences in blood pressure or serum lipids. In the OHP, % body fat mass was significantly correlated with systolic blood pressure (P < 0.05), triglyceride (P < 0.01) and apolipoprotein B (P < 0.01), not observed in controls. The serum IRI of the OHP, but not that of controls, was significantly correlated with systolic blood pressure (P < 0.05), serum triglyceride (P < 0.02) and apolipoprotein B (P < 0.05). Thus, a higher serum IRI level due to an increase in total body fat mass appears to be an inherited trait that contributes to the development of hypertension and dyslipidemia.

Oxidative stress (OS) plays a key role in the development of diabetes complications. Bilirubin is a potent antioxidant, and serum total bilirubin (TB) levels in the low-normal range are associated with diabetic retinopathy (1) and nephropathy (2) and are predictive of lower-limb amputation (3). However, the relationship between serum TB and diabetic neuropathy has not been reported. Due to constant exposure to OS (sunlight and ambient air) and poor antioxidant activity due to its avascularity, the cornea is vulnerable to OS (4). Diabetes increases the OS burden on the cornea. Corneal nerve fibers (CNFs) are composed of unmyelinated C-fibers, which are the first nerve fibers to undergo damage and subsequent repair. We examined the association between serum TB and CNF morphology in patients with uncontrolled type 2 diabetes (T2D). Cross-sectional data were collected from 384 …

Objective It is believed that oxidative stress, which is provoked in patients with diabetes, plays critical roles in the pathogenesis of myocardial infarction (MI). We simultaneously determined 5 relatively common genetic variants related to oxidative stress and evaluated the combined effect on MI. Methods We enrolled 3819 Japanese type 2 diabetic subjects (males 60.8%, 60.6±10.0 years old) and determined their genotypes regarding glutamate-cysteine ligase modifier subunit (GCLM) C-588T , manganese superoxide dismutase (SOD2) Val16Ala , endothelial nitric oxide synthase (NOS3) G894T , NAD(P)H oxidase p22phox (CYBA) C242T , and myeloperoxidase (MPO) G-463A polymorphisms. The diagnosis of the occurrence of MI was based on the clinical records, characteristic ECG changes, and the findings in coronary angiography and echocardiography. Results The single association analysis revealed that there were no statistically significant associations between each polymorphism and the prevalence of MI. Interestingly, the prevalence of MI was higher with the increase of the total number of 5 concomitant unfavorable "pro-oxidant alleles" in each subject ( p value for linear trend=0.018). Furthermore, a multiple logistic regression analysis showed that the number of pro-oxidant alleles was a risk factor for MI independently of conventional risk factors (odds ratio for 1-point increase in the number of pro-oxidant allele=1.16 with 95% CI 1.01–1.34, p =0.034). Conclusions Accumulation of gene polymorphisms related to oxidative stress is likely associated with the prevalence of MI in type 2 diabetic patients, suggesting that the combined information about these variants is useful to assess the risk of MI.

Connexin37 is one of the major connexins expressed in the endothelium, monocytes, and macrophages and plays important roles in atherogenesis (1). The COOH-terminus of this protein is a substrate for specific kinase or protein partners and thereby functions as a regulatory domain (2). Because the C-to-T substitution at nucleotide 1019 in the connexin37 gene causes a proline-to-serine substitution in the regulatory COOH-terminus, and affects its function, this polymorphism is likely associated with the pathogenesis of atherosclerosis. Indeed, this polymorphism has been shown to associate with the risk of coronary artery disease (3). In this study, we investigated whether this polymorphism is associated with an ankle-brachial blood pressure index (ABI), …

Lysosomal acidification is a key step of albumin reabsorption in proximal tubular epithelial cells (PTECs). This study was performed to examine the influence of chronic high glucose on lysosomal acidification in cultured PTECs. Porcine PTECs (LLC-PK 1 cells) were cultured in 16.7 mM (300 mg/dl) glucose (HG) alone or with 0.5 mM phlorizin for 24 weeks and subsequently for 12 weeks in 5.5 mM (100 mg/dl) glucose (NG). Chronic HG inhibited the fluorescein isothiocyanate (FITC)-albumin (A) uptake progressively, while phlorizin reversed the inhibition. NG for 12 weeks after HG normalized the uptake. The time-dependent uptake of FITC-A was inhibited by HG and bafilomycin A 1 (BafA 1 ) after 15 min and by 4,4′-diisothiocyanato-2,2′-disulfonic acid (DIDS) and N -ethyl- N -isopropyl-amiloride (EIPA) after 3 min. Cellular ATP was depleted by HG and restored by NG. Lysosomal pH, assessed by an acidotropic fluorescent probe, was alkalinized (pH 4.5–7.8) with 5.5–27.8 mM glucose and normalized by subsequent NG. BafA 1 alkalinized lysosomes, and the concentration required to 50% change for the pH and 50% inhibition of FITC-A uptake was similar. EIPA inhibited FITC-A uptake, but did not influence lysosomal pH. DIDS inhibited FITC-A uptake, and unexpectedly lowered lysosomal pH. Real time PCR showed that HG reduced the mRNA level for vacuolar H + -ATPase, but did not alter those of chloride channel-5 and Na + –H + -exchanger-3. In conclusion, the chronic HG inhibits albumin reabsorption by lysosomal alkalinization in PTECs, probably due to ATP depletion and down-regulation of vacuolar H + -ATPase.

In this study, we clarify that high glucose inhibits albumin uptake in cultured LLC-PK1 cells. LLC-PK1 cells cultured for 6 days with 5.5-27.8 mM D-glucose were challenged by fluorescein isothiocyanate (FITC)-conjugated human albumin (HA). FITC-HA binding and uptake were inhibited by5.5mM glucose (5.5 mM(P0.01) 11.0 mM(P0.05) 16.7 mM approximately= 27.8 mM). Analysis of FITC-HA binding and uptake at 5.5 and 16.7 mM D-glucose (high glucose, HG) showed decreased affinity (K(m) for binding: 35.5 mg/l versus 52.6 mg/l, K(m) for uptake; 41.3 mg/l versus 55.6 mg/l) and maximal velocity (B(max)--0.33 microg versus 0.27 microg/30 min/mg protein; U(max)--4.40 microg versus 3.48 microg/60 min/mg protein) at HG. A comparison of the time courses of FITC-HA binding and uptake at 5.5 mM glucose and at HG showed that HG suppressed them beyond 15 min (P0.005-0.001). Phlorizin (0.25 mM) completely reversed the HG-induced inhibition of FITC-HA binding and uptake. High glucose decreased mRNA of GLUT-1 and SGLT-1, but did not influence that of SGLT-2. The simultaneous presence of Vitamin E (10(-6)M), Vitamin C (10(-6)M) and reduced glutathione (0.25 mM) reversed the suppressed FITC-HA binding and uptake by HG, while any one or two of these molecules, and various inhibitors of advanced glycation end products, failed to do so. In conclusion, a high glucose milieu causes inhibition of albumin binding and uptake in proximal tubular cells by increasing metabolic oxidative stress through excessive glucose flux via the sodium glucose transporter.

Glomerular or tubular contribution for microalbuminuria was estimated by assessing albumin excretion rate (AER) in glomerular urine obtained by l -arginine infusion (AI) and glomerular filtration rate (GFR) in 20 non-insulin-dependent diabetes mellitus (NIDDM) patients before and after glycemic control, and in 19 age-matched controls. Glycemic control normalized AER during AI, while it decreased AER before AI, though it was still above normal. Glycemic control increased tubular reabsorption rate of albumin, but it was still less than normal. Tubular reabsorption rate of albumin declined in close relation with duration of diabetes mellitus, while AER in glomerular urine had no correlation with the duration. GFR had no correlation with AER before or during AI. In conclusion, impaired tubular reabsorption of albumin plays a key role for microalbuminuria in NIDDM.

Our objective was to determine the role of increased glomerular clearance (GC) or reduced tubular reabsorption (TR) of transferrin in producing microtransferrinuria. An infusion of l -arginine was used to inhibit TR of transferrin, permitting the determination of both GC and TR of transferrin in 64 patients with non-insulin-dependent diabetes mellitus (NIDDM), with or without microtransferrinuria. Thirty-one healthy volunteers served as control subjects. The GC of transferrin in NIDDM patients with microtransferrinuria did not differ significantly from that in healthy controls or in NIDDM patients with normal transferrin excretion rates (TfER). No correlation was found between TfER and GC of transferrin in any group of the subjects. However, the TR of transferrin was inversely correlated with TfER in healthy controls and in the NIDDM patients, with or without microtransferrinuria. When transferrin absorption was plotted against the filtered load of transferrin, the regression lines for the three subject groups were parallel. The regression line for NIDDM patients with microtransferrinuria was shifted to the right of those for healthy controls and NIDDM patients with a normal TfER. These findings suggest that microtransferrinuria in patients with NIDDM is caused by the impaired tubular reabsorption of transferrin.

The alteration in glomerular clearance (GC) and tubular reabsorption (TR) of IgG subclass (IgG1 and IgG4, same molecular weight, but differing charge) was investigated in 14 microalbuminuric patients with non-insulin-dependent diabetes mellitus (NIDDM) by inhibiting TR of proteins by l -arginine infusion; 16 healthy volunteers served as controls. In healthy volunteers, GC was in the following order: IgG1 > IgG4 ≥ albumin. In microalbuminuric patients, as compared with control, GC of albumin was increased significantly; the GC of IgG4 was twice that of control, while GC of IgG1 decreased reciprocally. The TR in controls was in the following order; albumin > IgG1 > IgG4. In microalbuminuric patients, TR of albumin was significantly reduced, TR of IgG1 was moderately reduced and that of IgG4 remained at same level as controls. These observations suggest that the enhancement of GC of albumin in NIDDM was produced by an impaired glomerular anionic charge barrier, while the mechanism for the reduction in TR of albumin was attributable to other undefined mechanisms.

It is well-known that oxidative stress plays critical roles in the pathogenesis of atherosclerotic diseases. In this study, we examined the association between four common genetic variants related to oxidative stress (glutamate-cysteine ligase modifier subunit (GCLM) C-588T, myeloperoxidase G-463A, human paraoxonase-1 Gln192Arg, and NADPH oxidase p22phox C242T) and the prevalence of myocardial infarction in type 2 diabetic patients. All type 2 diabetic patients who periodically attended the outpatient diabetes clinics of five participating hospitals were asked to participate in this study. Subjects who were eligible had type 2 diabetes, diagnosed by diabetologists based on World Health Organization criteria, and were aged ≥50 years. A total of 2,561 type 2 diabetic subjects were included: men, 62%; age, mean ± SD 60.9 …

The differences in deranged renal hemodynamics in non-insulin-dependent diabetes mellitus (NIDDM) and insulin-dependent diabetes mellitus (IDDM) have never been fully investigated. Whether or not autoregulatory mechanism of renal hemodynamics in NIDDM and IDDM is preserved remains to be clarified. In the present study we directly compared renal hemodynamics and its autoregulatory function in the early stage of NIDDM and IDDM before and after short-term glycemic control. Before glycemic control, mildly exaggerated glomerular filtration rate (GFR), subnormal renal plasma flow (RPF), and elevated filtration fraction (FF) were found in NIDDM as well as in IDDM; but there were no differences in these parameters of renal hemodynamics between the two types of diabetics. Glycemic control decreased GFR, whereas it did not alter RPF, resulting in normalization of FF in NIDDM and in IDDM. Before glycemic control, mean blood pressure was significantly correlated with GFR, but was not correlated after glycemic control in either type of diabetes. In conclusion, hyperglycemia induced glomerular hyperfiltration evenly and disturbed autoregulation of renal hemodynamics in NIDDM and in IDDM.

Methods have been developed for measuring the intracellular water space (ICS) in cultured diploid fibroblast monolayers. The values obtained have been compared to similar measurements of ICS in suspended fibroblasts using an oil spin method. Markers commonly used for measurement of total water space (TWS) ([3H]H2O, [14C]urea) and extracellular space (ECS) ([3H]inulin, [14C]L-glucose, [3H]sucrose, [3H]D-mannitol) were investigated for use in cell monolayers. Monolayer incubations were terminated by rapidly rinsing the culture dishes three times with ice cold buffer. The distribution volume of the TWS marker [14C]urea plateaued at 10 to 15 min and was independent of urea concentration. [3H]H2O was not a suitable marker for measuring total water space in cell monolayers because of rapid loss of intracellular label during rinsing. Intracellular space was calculated by subtracting [3H]sucrose space (5 min) from [14C]urea space (20 min) after incubation of fibroblasts with both markers. Values obtained for ICS (mean +/- SE: microliter/10(6) cells) of fibroblasts from two cell lines measured in monolayer (1.74 +/- 0.11 and 1.60 +/- 0.10) and in suspension (1.88 +/- 0.07 and 1.78 +/- 0.11) was approximately 10% smaller than the values for cell size (2.01 and 2.22) obtained from Coulter Counter sizing. Thus, the methods developed for measurement of ICS in monolayer fibroblasts yield data comparable to those obtained with the more standard oil spin method. Furthermore, the methods can be applied to measurement of ICS in other types of adherent cells.