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Aims/Introduction Diabetic kidney disease is characterized by increased albuminuria and/or a reduced glomerular filtration rate (GFR). We analyzed secular changes in the prevalence of albuminuria and reduced estimated GFR (eGFR) in Japanese patients with type 2 diabetes, and identified factors associated with these changes. Materials and Methods Using 1996, 2001, 2006 and 2014 cohort data from the Japanese serial cross‐sectional studies conducted at Shiga University of Medical Science, secular changes in the prevalence of diabetic kidney disease (albuminuria and/or reduced eGFR), patient characteristics and their associations were analyzed. Results The prevalence of microalbuminuria and macroalbuminuria decreased over time, whereas the prevalence of moderately reduced eGFR (30–60 mL/min/1.73 m2) and severely reduced eGFR (

IntroductionGiven an increasing use of dipeptidyl peptidase-4 (DPP-4) inhibitors to treat patients with type 2 diabetes mellitus in the real-world setting, we conducted a prospective observational study (Japan-based Clinical Research Network for Diabetes Registry: J-BRAND Registry) to elucidate the safety and efficacy profile of long-term usage of alogliptin.Research design and methodsWe registered 5969 patients from April 2012 through September 2014, who started receiving alogliptin (group A) or other classes of oral hypoglycemic agents (OHAs; group B), and were followed for 3 years at 239 sites nationwide. Safety was the primary outcome. Symptomatic hypoglycemia, pancreatitis, skin disorders of non-extrinsic origin, severe infections, and cancer were collected as major adverse events (AEs). Efficacy assessment was the secondary outcome and included changes in hemoglobin A1c (HbA1c), fasting blood glucose, fasting insulin and urinary albumin.ResultsOf the registered, 5150 (group A: 3395 and group B: 1755) and 5096 (3358 and 1738) were included for safety and efficacy analysis, respectively. Group A patients mostly (>90%) continued to use alogliptin. In group B, biguanides were the primary agents, while DPP-4 inhibitors were added in up to ~36% of patients. The overall incidence of AEs was similar between the two groups (42.7% vs 42.2%). Kaplan-Meier analysis revealed the incidence of cancer was significantly higher in group A than in group B (7.4% vs 4.8%, p=0.040), while no significant incidence difference was observed in the individual cancer. Multivariate Cox regression analysis revealed that the imbalanced patient distribution (more elderly patients in group A than in group B), but not alogliptin usage per se, contributed to cancer development. The incidence of other major AE categories was with no between-group difference. Between-group difference was not detected, either, in the incidence of microvascular and macrovascular complications. HbA1c and fasting glucose decreased significantly at the 0.5-year visit and nearly plateaued thereafter in both groups.ConclusionsAlogliptin as a representative of DPP-4 inhibitors was safe and durably efficacious when used alone or with other OHAs for patients with type 2 diabetes in the real world setting.

Background Neurofibromatosis type 1 is characterized by multiple café au lait spots and cutaneous and plexiform neurofibromas, and is one of the most common autosomal dominant hereditary disorders caused by mutations of the neurofibromatosis type 1 tumor suppressor gene. Osteomalacia in neurofibromatosis type 1 is very rare and is characterized by later onset in adulthood. In humans, fibroblast growth factor 23, which is a causative factor of tumor-induced osteomalacia, is not only a paracrine and autocrine factor, but is also a physiological regulator of phosphate balance in normal serum. Case presentation Our patient was a 65-year-old Japanese woman whose neurofibromas began to appear when she was in elementary school. At age 28, she was diagnosed as having neurofibromatosis type 1. A spinal compression fracture and multiple rib fractures were identified in 2012 and 2017, respectively. Her laboratory findings revealed hypophosphatemia due to renal phosphate wasting and a high serum level of fibroblast growth factor 23. Neurofibromas located on the surface of her right forearm and left upper arm, in which a slight abnormal accumulation of tracers was observed on 111indium-pentetreotide scintigraphy, were surgically removed, but there was no improvement in hypophosphatemia or serum fibroblast growth factor 23 after surgery. Therefore, we administered eldecalcitol, which also failed to produce improvement in abnormal data. Subsequent combination with dibasic calcium phosphate hydrate led to improvement in some of the abnormalities, including hypophosphatemia. Immunohistochemical staining using anti-human fibroblast growth factor 23 antibody revealed slightly positive results, however, only one out of three amplifications of the fibroblast growth factor 23 gene was observed by real-time polymerase chain reaction, and no clear fibroblast growth factor 23 gene expression in the resected neurofibromas could be confirmed. Conclusions We here describe a first rare case of a 65-year-old woman with neurofibromatosis type 1 associated with hypophosphatemic osteomalacia in which a high serum fibroblast growth factor 23 level was confirmed.

BackgroundThe prevalence of childhood-onset growth hormone (GH) deficiency (GHD) is estimated to be approximately 1 in 5000 or more, with the cause unknown in most cases (idiopathic isolated GHD). However, additional disorders of secretion of other pituitary hormones reportedly develop over time, with a frequency of 2–94% (median, 16%). Furthermore, median times to development of other anterior pituitary hormone deficiencies have been reported to be 6.4–9.4 years. On the other hand, adult patients affected by childhood-onset GHD reportedly develop impaired ventilation function due to reduced lung volumes and respiratory pressures, probably due to reductions in respiratory muscle strength. In addition, GH is known to play a role in stimulating the glomerular filtration rate (GFR), and the estimated GFR (eGFR) is decreased in patients with GHD.Case presentationThis case involved a 65-year-old woman. Her short stature had been identified at around 3 years of age, but no effective treatments had been provided. The patient was mostly amenorrheic, and hair loss became apparent in her late 30s. She developed hyperuricemia, dyslipidemia, and hypertension at 45 years of age. In addition, the patient was diagnosed with hypothyroidism at 50 years of age. At 58 years of age, endocrinological examination showed impaired secretion of thyroid-stimulating hormone, luteinizing hormone/follicle-stimulating hormone, and growth hormone, and magnetic resonance imaging showed an empty sella turcica. However, secretion ability of adrenocorticotropic hormone was retained. At 63 years of age, respiratory function tests confirmed a markedly restricted ventilation disorder (vital capacity, 0.54 L; percentage predicted vital capacity, 26.9%). Renal function had also decreased (eGFR, 25.0 mL/min/1.73 m2). Furthermore, she was diagnosed with hypothalamic secondary hypoadrenocorticism. The patient developed CO2narcosis at 65 years of age, and noninvasive positive pressure ventilation was started.ConclusionsThe rare case of a 65-year-old woman with childhood-onset GHD with panhypopituitarism, including late-onset secondary hypoadrenocorticism in her 60s, associated with severely impaired respiratory function and renal dysfunction, was reported. In GHD patients with risk factors for progression from isolated GHD to combined pituitary hormone deficiency, such as empty sella turcica, lifelong endocrinological monitoring may be important.

Renal dysfunction in addition to diabetes is a serious risk factor for cardiovascular events. We hypothesized that some of the changes in gene expression in blood cells cause renal dysfunction and macrovascular disease through impaired endothelial function. This study aimed to define which changes in gene expression in peripheral blood mononuclear cells (PBMCs) are related to renal function parameters and endothelial function of large arteries in patients with type 2 diabetes mellitus (T2DM).We recruited 95 patients with T2DM. After matching for gender, age, BMI and HbA1c levels, the patient cohort included 42 with normoalbuminuria, 28 with microalbuminuria, and 25 with macroalbuminuria. All patients in the three groups were assessed for urinary albumin to creatinine ratio (ACR), estimated glomerular filtration rate (eGFR), flow-mediated dilatation (FMD), and mRNA expression in PBMCs.The mRNA expression of platelet activating factor receptor (PAFR) differed most markedly between the three groups and was significantly higher in the macroalbuminuric group (p 0.001 vs. normoalbuminuric group; p 0.05 vs. microalbuminuric group). PAFR mRNA expression significantly correlated with log transformed ACR (ρ = 0.424, p 0.001) but not eGFR. PAFR mRNA expression also had a significant negative correlation with FMD (ρ = -0.379, p 0.001). Furthermore, the prevalence of macrovascular complications, particularly stroke, was significantly higher in patients with elevated PAFR mRNA expression in PBMCs.PAFR overexpression in PBMCs may link diabetic nephropathy to macroangiopathy through impairment of endothelial function in patients with T2DM.

Aim: Elevated level of serum triglyceride (TG) is a characteristic of type 2 diabetes. We evaluated the clinical significance of intervention for the serum TG levels in the fasting and postprandial states in patients with type 2 diabetes. Methods: Fifty patients with type 2 diabetes, treated with statins, were selected and divided into two groups. One group was treated with a combination of fenofibrate and ezetimibe (F/E group) and the other group with statins (statin group) for 12 weeks. The lipoprotein profile of both groups was compared using high-performance liquid chromatography, and the vascular function was assessed using flow-mediated dilation (FMD) at the forearm. Results: The levels of very low-density lipoprotein (VLDL) cholesterol, malondialdehyde low-density lipoprotein (MDA-LDL), total TG, chylomicron-TG, VLDL-TG, and HDL-TG decreased in the F/E group, whereas those of HDL cholesterol increased. Furthermore, the peak particle size of LDL increased, but that of HDL decreased in the F/E group. The combination treatment significantly improved the FMD. The change in the cholesterol level in a very small fraction of HDL was a significant independent predictor for determining the improvement of FMD (p < 0.01). Conclusions: Compared with the treatment with statins, the treatment with the combination of fenofibrate and ezetimibe effectively controlled the LDL cholesterol and TG levels, increased the HDL cholesterol level, especially in its small fraction, and improved vascular function of patients with type 2 diabetes.

Glucose-stimulated insulin secretion (GSIS) by pancreatic β cells is biphasic. However, the physiological significance of biphasic GSIS and its relationship to diabetes are not yet fully understood. This study demonstrated that impaired first-phase GSIS follows fasting, leading to increased blood glucose levels and brain glucose distribution in humans. Animal experiments to determine a possible network between the brain and β cells revealed that fasting-dependent hyperactivation of AMP-activated protein kinase in the hypothalamus inhibited first-phase GSIS by stimulating the α-adrenergic pancreatic nerve. Furthermore, abnormal excitability of this brain-β cell neural axis was involved in diabetes-related impairment of first-phase GSIS in diabetic animals. Finally, pancreatic denervation improved first-phase GSIS and glucose tolerance and ameliorated severe diabetes by preventing β cell loss in diabetic animals. These results indicate that impaired first-phase GSIS is critical for brain distribution of dietary glucose after fasting. Furthermore, β cells in individuals with diabetes mistakenly sense that they are under conditions that mimic prolonged fasting. The present study provides additional insight into both β cell physiology and the pathogenesis of β cell dysfunction in type 2 diabetes., Highlights • Fasting-induced hypothalamic AMPK activation inhibited first-phase GSIS by stimulating the α-adrenergic nerve. • The brain-pancreas neural axis was involved in β cell dysfunction and glucose intolerance in diabetes. • Pancreatic denervation improved first-phase GSIS, glucose tolerance and β cell survival in type 2 diabetic animals. Glucose-stimulated insulin secretion (GSIS) from pancreatic β cells is biphasic. Furthermore, first-phase GSIS is inhibited in type 2 diabetes. This study revealed that fasting reduced first-phase GSIS by signaling via the brain-pancreatic β cell neural axis, which is essential for maintaining glucose supply to the brain at re-feeding after fasting. Abnormal excitability of this neural axis was also associated with impaired first-phase GSIS in type 2 diabetes. Surgical pancreatic denervation improved diabetes in an animal study. The present data reveal that diabetic β cells exist under conditions that mimic starvation and provide a therapeutic potency of pancreatic denervation against diabetes., Graphical Abstract Image 2

The beneficial effects of fish and n-3 polyunsaturated fatty acids (PUFAs) consumption on atherosclerosis have been reported in numerous epidemiological studies. However, to the best of our knowledge, the effects of a fish-based diet intervention on endothelial function have not been investigated. Therefore, we studied these effects in postmenopausal women with type 2 diabetes mellitus (T2DM).Twenty-three postmenopausal women with T2DM were assigned to two four-week periods of either a fish-based diet (n-3 PUFAs ≧ 3.0 g/day) or a control diet in a randomized crossover design. Endothelial function was measured with reactive hyperemia using strain-gauge plethysmography and compared with the serum levels of fatty acids and their metabolites. Endothelial function was determined with peak forearm blood flow (Peak), duration of reactive hyperemia (Duration) and flow debt repayment (FDR).A fish-based dietary intervention improved Peak by 63.7%, Duration by 27.9% and FDR by 70.7%, compared to the control diet. Serum n-3 PUFA levels increased after the fish-based diet period and decreased after the control diet, compared with the baseline (1.49 vs. 0.97 vs. 1.19 mmol/l, p0.0001). There was no correlation between serum n-3 PUFA levels and endothelial function. An increased ratio of epoxyeicosatrienoic acid/dihydroxyeicosatrienoic acid was observed after a fish-based diet intervention, possibly due to the inhibition of the activity of soluble epoxide hydrolase.A fish-based dietary intervention improves endothelial function in postmenopausal women with T2DM. Dissociation between the serum n-3 PUFA concentration and endothelial function suggests that the other factors may contribute to this phenomenon.

Periodontal disease is related to aging, smoking habits, diabetes mellitus, and systemic inflammation. However, there remains limited evidence about causality from intervention studies. An effective diet for prevention of periodontal disease has not been well established. The current study was an intervention study examining the effects of a high-fiber, low-fat diet on periodontal disease markers in high-risk subjects. Forty-seven volunteers were interviewed for recruitment into the study. Twenty-one volunteers with a body mass index of at least 25.0 kg/m(2) or with impaired glucose tolerance were enrolled in the study. After a 2- to 3-week run-in period, subjects were provided with a test meal consisting of high fiber and low fat (30 kcal/kg of ideal body weight) 3 times a day for 8 weeks and followed by a regular diet for 24 weeks. Four hundred twenty-five teeth from 17 subjects were analyzed. Periodontal disease markers assessed as probing depth (2.28 vs 2.21 vs 2.13 mm; P < .0001), clinical attachment loss (6.11 vs 6.06 vs 5.98 mm; P < .0001), and bleeding on probing (16.2 vs 13.2 vs 14.6 %; P = .005) showed significant reductions after the test-meal period, and these improvements persisted until the follow-up period. Body weight (P < .0001), HbA1c (P < .0001), and high-sensitivity C-reactive protein (P = .038) levels showed improvement after the test-meal period; they returned to baseline levels after the follow-up period. In conclusion, treatment with a high-fiber, low-fat diet for 8 weeks effectively improved periodontal disease markers as well as metabolic profiles, at least in part, by effects other than the reduction of total energy intake.

Objective:To elucidate early skin denervation in hereditary transthyretin (TTR) amyloidosis and iatrogenic TTR amyloidosis.Methods:We investigated intraepidermal nerve fiber density (IENFD) and clinical findings in 32 patients with hereditary TTR amyloidosis, 11 asymptomatic mutation carriers, 6 patients with iatrogenic TTR amyloidosis, and 23 healthy volunteers.Results:IENFD values were reduced in patients with the V30M mutation (1.9 ± 2.1 per 1 mm), patients with non-V30M mutations (5.8 ± 3.2 per 1 mm), and patients with iatrogenic TTR amyloidosis (3.5 ± 1.8 per 1 mm) compared with healthy volunteers (11.8 ± 3.2 per 1 mm) (p < 0.01). Skin denervation also occurred, even in presymptomatic V30M mutation carriers (5.0 ± 2.2 per 1 mm). The IENFD was correlated with disease duration (ρ = −0.533, p = 0.002) and various peripheral neuropathy parameters such as sensory impairment in the Kumamoto clinical score (ρ = −0.575, p = 0.001), heat-pain detection threshold (ρ = −0.704, p < 0.001), and sural sensory nerve action potential (ρ = 0.481, p = 0.005). TTR amyloid deposits frequently occurred in connective tissues and vessels of the dermal reticular layer in patients with hereditary TTR amyloidosis and those with iatrogenic TTR amyloidosis.Conclusions:Patients with hereditary TTR amyloidosis and those with iatrogenic TTR amyloidosis may show early skin denervation even in the presymptomatic stage. IENFD may thus be useful for early diagnosis and may serve as a biomarker in clinical trials for hereditary and iatrogenic TTR amyloidosis.

Background & Aims A fiber-rich diet has a cardioprotective effect, but the mechanism for this remains unclear. We hypothesized that a fiber-rich diet with brown rice improves endothelial function in patients with type 2 diabetes mellitus. Methods Twenty-eight patients with type 2 diabetes mellitus at a single general hospital in Japan were randomly assigned to a brown rice (n = 14) or white rice (n = 14) diet and were followed for 8 weeks. The primary outcome was changes in endothelial function determined from flow debt repayment by reactive hyperemia using strain-gauge plethysmography in the fasting state. Secondary outcomes were changes in HbA1c, postprandial glucose excursions, and markers of oxidative stress and inflammation. The area under the curve for glucose after ingesting 250 kcal of assigned rice was compared between baseline (T0) and at the end of the intervention (T1) to estimate glucose excursions in each group. Results Improvement in endothelial function, assessed by fasting flow debt repayment (20.4% vs. −5.8%, p = 0.004), was significantly greater in the brown rice diet group than the white rice diet group, although the between-group difference in change of fiber intake was small (5.6 g/day vs. −1.2 g/day, p

Aim and methods To assess the usefulness of the diagnostic and staging criteria for diabetic polyneuropathy (DP) by the Diabetic Neuropathy Study Group in Japan (DNSGJ) we examined clinical features, intraepidermal nerve fiber densities (IENFD) and nerve conduction studies (NCS) and coefficient of variation of the R-R intervals (CVR-R) in 44 patients with diabetes. Results The patients were classified into stage I ( n = 20), II ( n = 6), III + IV ( n = 12), and V ( n = 6) according to the staging criteria by DNSGJ. IENFD decreased as stages progressed (13.8 ± 7.1 fiber/mm in stage I to 0.8 ± 1.3 fiber/mm in stage V). Compound motor and sensory action potential and motor and sensory nerve conduction velocity decreased with progressing stage. F-wave latency prolonged as stages progressed. CVR-R decreased with progressing stage (4.41% ± 2.65% in stage I to 1.33% ± 0.57% in stage V). IENFD correlated with the various parameters of NCS ( r = 0.378–0.636, p r = 0.399, p = 0.007). Conclusions Clinical staging for DP by DNSGJ reflects the results of small and large fiber neuropathy.

Background:Smoking is an overwhelming, but preventable, risk factor for cardiovascular diseases (CVD), although smoking prevalence remains high in developed and developing countries in East Asia., Methods and results:In a population-based sample of 1019 Japanese men aged 40 to 79 years, without CVD, we examined cross-sectional associations of smoking status, cumulative pack-years, daily consumption, and time since cessation, with subclinical atherosclerosis at 4 anatomically distinct vascular beds, including coronary artery calcification, carotid intima-media thickness (CIMT) and plaque, aortic artery calcification (AoAC), and ankle-brachial index. Current, former, and never smoking were present in 32.3%, 50.0%, and 17.7%, respectively. Compared to never smokers, current smokers had significantly higher risks of subclinical atherosclerosis in all 4 circulations (eg, odds ratios for coronary artery calcification >0, 1.79 [95% CIs, 1.16-2.79]; CIMT >1.0 mm, 1.88 [1.02-3.47]; AoAC >0, 4.29 [2.30-7.97]; and ankle-brachial index 1.0 mm, 1.94 [1.13-3.34]; and AoAC >0, 2.55 [1.45-4.49]). Dose-response relationships of pack-years and daily consumption, particularly with CIMT, carotid plaque, AoAC, and ankle-brachial index, were observed among both current and former smokers, and even a small amount of pack-years or daily consumption among current smokers was associated with coronary artery calcification and AoAC, whereas time since cessation among former smokers was linearly associated with lower burdens of all atherosclerotic indices., Conclusions:Cigarette smoking was strongly associated with subclinical atherosclerosis in multiple vascular beds in Japanese men, and these associations attenuated with time since cessation.

The aim of this study was to evaluate the ability of the growth hormone-releasing peptide-2 (GHRP-2) test to clinically diagnose hypothalamo-pituitary-adrenal (HPA) axis failure. We performed an insulin tolerance test (ITT), CRH stimulation test, and GHRP-2 test on 47 patients suspected of having a hypothalamo-pituitary disorder. Patients with pituitary disorders had significantly lower ACTH responses to the GHRP-2 test compared to patients with hypothalamic disorders and the control group. In contrast, peak cortisol levels in response to the GHRP-2 test were significantly lower in both hypothalamic and pituitary disorder cases compared with the control group. Assignment of a cut-off value of 11.6 μg/dL for the peak serum cortisol level demonstrated that the GHRP-2 test was able to predict secondary hypoadrenalism with 88.9% specificity and 89.7% sensitivity. The responses of ACTH and cortisol to the GHRP-2 test had no correlation to the CRH test, suggesting the involvement of a different mechanism of ACTH secretion. These results indicate that the GHRP-2 test may induce ACTH secretion from the pituitary gland through direct stimulation. Although the GHRP-2 test does not have the same predictive value as the insulin tolerance test (ITT), it has similar diagnostic potential as the CRH stimulation test for evaluating HPA axis failure.

ObjectiveThe glomerular filtration rate (GFR) is increased in patients with active acromegaly. The aim of this study is to elucidate whether renal function deteriorates in patients with acromegaly and whether this deterioration is reversible after surgical remission.Design/methodsA case–control study of 48 acromegalic patients who were surgically cured (cases) and 48 patients with nonfunctioning pituitary adenomas (NFomas, controls) was conducted. We performed clinical and biochemical examinations before surgery and 3months post-surgery. The GFR of each patient was estimated (estimated GFR, eGFR) using their serum creatinine, age, sex, and body surface area, and postoperative changes in the eGFR were assessed.ResultsThe preoperative eGFR was significantly higher in patients with acromegaly than in those with NFoma (99.8 vs 75.1mL/min respectively,PPP=0.12). Among the acromegalic patients, the postoperative decreases in the eGFR were more prominent in patients with a preoperatively high or normal vs low eGFR.ConclusionsOur data demonstrated a significant post-surgical eGFR decrease in patients with acromegaly, but not in patients with NFomas. This change in the eGFR was reversible in acromegalic patients with a high/normal preoperative eGFR, but not in those with a low preoperative eGFR. This suggests that the reversible pathophysiological change in some patients is functional but not organic.

Several lines of epidemical evidence have shown that type 2 diabetes is the most important risk factor for cardiovascular diseases (CVD). It has been shown that the risk of primary prevention of CVD in patients with diabetes is equal to that of the secondary prevention in general population. In this manuscript, recent reports on the cardiac tests to detect the cardiovascular lesions will be reviewed. The data suggest that MDCT is a promising test even in the patients with diabetes. Furthermore, recent evidence of the treatment of diabetes with insulin or the drugs available recently such as DPP-4 inhibitors and SGLT-2 inhibitors will be reviewed.

Free fatty acid (FFA)-bound albumin, which is filtrated through the glomeruli and reabsorbed into proximal tubular cells, is one of the crucial mediators of tubular damage in proteinuric kidney disease. In this study, we examined the role of each kind of FFA on renal tubular damage in vitro and tried to identify its molecular mechanism. In cultured proximal tubular cells, a saturated fatty acid, palmiate, increased the expression of monocyte chemoattractant protein-1 (MCP-1), but this effect was abrogated by co-incubation of monounsaturated fatty acid, oleate, or ω-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA). Palmitate led to intracellular accumulation of diacylglycerol (DAG) and subsequent activation of protein kinase C protein family. Among the several PKC inhibitors, rottlerin, a PKCθ inhibitor, prevented palmitate-induced MCP-1 expression via inactivation of NFB pathway. Overexpression of dominant-negative PKCθ also inhibited palmitate-induced activation of MCP-1 promoter. Furthermore, palmitate enhanced PKCθ-dependent mitochondrial apoptosis, which was also prevented by co-incubation with oleate or EPA through restoration of pro-survival Akt pathway. Moreover, oleate and EPA inhibited palmitate-induced PKCθ activation through the conversion of intracellular DAG to triglyceride with the restoration of diacylglycerol acyltransferase 2 expression. These results suggest that oleate and EPA have protective effects against the palmitate-induced renal tubular cell damage by inhibiting PKCθ activation.

OBJECTIVE Cross-sectional studies have reported increased levels of urinary type IV collagen in diabetic patients with progression of diabetic nephropathy. The aim of this study was to determine the role of urinary type IV collagen in predicting development and progression of early diabetic nephropathy and deterioration of renal function in a longitudinal study. RESEARCH DESIGN AND METHODS Japanese patients with type 2 diabetes (n = 254, 185 with normoalbuminuria and 69 with microalbuminuria) were enrolled in an observational follow-up study. The associations of urinary type IV collagen with progression of nephropathy and annual decline in estimated glomerular filtration rate (eGFR) were evaluated. RESULTS At baseline, urinary type IV collagen levels were higher in patients with microalbuminuria than in those with normoalbuminuria and correlated with urinary β2-microglobulin (β = 0.57, P < 0.001), diastolic blood pressure (β = 0.15, P < 0.01), eGFR (β = 0.15, P < 0.01), and urinary albumin excretion rate (β = 0.13, P = 0.01) as determined by multivariate regression analysis. In the follow-up study (median duration 8 years), urinary type IV collagen level at baseline was not associated with progression to a higher stage of diabetic nephropathy. However, the level of urinary type IV collagen inversely correlated with the annual decline in eGFR (γ = −0.34, P < 0.001). Multivariate regression analysis identified urinary type IV collagen, eGFR at baseline, and hypertension as factors associated with the annual decline in eGFR. CONCLUSIONS Our results indicate that high urinary excretion of type IV collagen is associated with deterioration of renal function in type 2 diabetic patients without overt proteinuria.

To determine the potential role of the transcriptional factor-activating enhancer-binding protein-2beta (TFAP2B) in the regulation of expression of adipokines, adiponectin, leptin, and interleukin-6 (IL-6) in vivo, we quantified the mRNA expression levels of these adipokines and TFAP2B in visceral (omental) and abdominal subcutaneous adipose tissues of 66 individuals with variable degree of adiposity and studied their correlations with BMI and their plasma concentrations. We found that BMI correlated negatively with plasma adiponectin levels and positively with those of leptin. Adiponection mRNA expression in subcutaneous fat correlated negatively with BMI, whereas leptin mRNA levels in the omentum correlated with plasma leptin levels and BMI. In contrast, IL-6 mRNA levels in subcutaneous and omental fat did not correlate with BMI. IL-6 mRNA levels in the omental fat correlated with plasma IL-6 levels. Whereas TFAP2B mRNA expression did not correlate with BMI, it correlated negatively with adiponectin expression in the subcutaneous adipose tissue. Furthermore, TFAP2B mRNA expression correlated negatively with leptin and positively with IL-6 expression in both subcutaneous and omental adipose tissues. These relationships are consistent with our in vitro observations and indicate that TFAP2B seems to regulate the expression of various adipokines in vivo.

We have previously reported an association between the activator protein-2beta (AP-2beta) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with a disease-susceptible allele of AP-2beta showed stronger AP-2beta expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta led to lipid accumulation and induced insulin resistance in 3T3-L1 adipocytes.We found that overexpression of AP-2beta in 3T3-L1 adipocytes decreased the promoter activity of leptin, and subsequently decreased both messenger RNA (mRNA) and protein expression and secretion. Furthermore, knockdown of endogenous AP-2beta by RNA-interference increased mRNA and protein expression of leptin. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed specific binding of AP-2beta to leptin promoter regions in vitro and in vivo. In addition, site-directed mutagenesis of the AP-2-binding site located between position +34 and +42 relative to the transcription start site abolished the inhibitory effect of AP-2beta. Our results clearly showed that AP-2beta directly inhibited insulin-sensitizing hormone leptin expression by binding to its promoter.AP-2beta modulated the expression of leptin through direct interaction with its promoter region.

Adiponectin has insulin-sensitizing, anti-atherogenic, and anti-inflammatory properties, and researchers have recently reported that omega-3 polyunsaturated fatty acid (PUFA) can increase the serum adiponectin concentration, suggesting that dietary factors, such as fish intake, may have an influence on the serum adiponectin concentration. In general, Japanese subjects consume twice as much fish as people in other countries. We hypothesized that incremental change in serum omega-3 PUFA levels by fish intake is an important regulator of serum adiponectin even in Japanese subjects. The aim of this study was to explore the relationship among fish consumption, serum omega-3 PUFA, such as eicosapentaenoic acid (EPA), levels, and serum adiponectin levels.We recruited 17 healthy Japanese volunteers (seven men and 10 women) for an 8-week fish-diet intervention (omega-3 PUFA 3.0 g/day) without affecting total energy intake, and measured serum adiponectin concentration and fatty acid profiles.Fish-diet intervention significantly increased the serum adiponectin concentration in women (from 13.5+/-4.6 to 15.8+/-5.2 microg/mL, p0.01) but not in men (from 8.7+/-2.8 to 8.7+/-2.5 microg/mL). Serum omega-3 PUFA increased more in female subjects than male subjects after the fish-diet intervention (57.3+/-86.6 vs 150.9+/-46.7 microg/mL, p=0.011), suggesting that changes in omega-3 PUFA concentration may explain the different response between sexes.A fish-based diet intervention increased the serum adiponectin concentration in young, non-obese, healthy Japanese female subjects. The increment in serum omega-3 PUFA may regulate the serum adiponectin concentration.

Among Asians, including Japanese, obesity is related to dyslipidemia and insulin resistance at a lower level of body mass index (BMI) compared with non-Hispanic whites (NHW). We hypothesize that this ethnic difference in the relationship between BMI and metabolic risks is partly associated with the ethnic difference in fat distribution, namely, liver fat as well as visceral adipose tissue. To compare liver fat content among Japanese vs NHW men, regional computed tomographic images were taken to measure liver computed tomographic density in population-based samples of 313 Japanese and 288 NHW men aged 40 to 49 years, along with the assessment of metabolic parameters. Liver fat content was higher in Japanese than NHW men (liver to spleen attenuation ratio [lower value means higher liver fat content]: 1.01 +/- 0.16 vs 1.07 +/- 0.15, respectively; P < .01), despite a lower mean BMI in Japanese men (BMI: 23.6 +/- 2.9 vs 27.8 +/- 4.2 kg/m(2), P < .01). Moreover, Japanese men had a greater disposition for fatty liver with a small increase in BMI than NHW (P < .01), whereas both groups had a similar relationship between visceral adipose tissue and BMI. In both groups, liver fat content correlated with triglycerides, homeostasis model assessment of insulin resistance, and C-reactive protein. Liver fat content is higher among Japanese than NHW; and this ethnic difference becomes more robust with a small increase in BMI, suggesting that fatty liver is a sensitive marker for the failure of the adipose tissue to expand to accommodate an increased energy influx, and is associated with similar metabolic risk in Japanese despite lower BMI compared with NHW men.

Inflammatory signaling via Toll-like receptor 4 (TLR4) has been shown to facilitate atherogenesis. Recent lines of evidence show that saturated fatty acids (SFAs) induce the inflammatory response via the TLR4 pathway in macrophages and adipocytes. The aims of this study are to confirm the role of SFAs in TLR4-mediated inflammatory signaling in vascular cells and to propose soy phosphatidylcholine (SPC) as an effective inhibitor against TLR4-mediated agonists. SFAs such as palmitate and stearate increased the expression and secretion of MCP-1 in human umbilical vein endothelial cells (HUVECs) and rat vascular smooth muscle cells (VSMCs). SFAs up-regulated the activity of MCP-1 promoter through the activation of NF-kappaB. Knockdown of TLR4 using siRNA diminished the SFA-induced MCP-1 expression in HUVECs and rat VSMCs, while PKC or ceramide signal inhibitor did not inhibit the expression. Furthermore, we found that SPC effectively inhibited the MCP-1 expression induced by palmitate or LPS in a dose-dependent manner. However, SPC did not inhibit the mRNA expression of MCP-1 induced by cytokines such as TNF-alpha and IL-1beta, or by agonists binding to TLRs other than TLR4. In addition, SPC did not affect the activity of LPS assessed by clotting activity of the Limulus amoebocyte lysate. These results clearly show that SPC specifically inhibits the inflammatory responses induced by the TLR4-dependent signal. In conclusion, we have demonstrated a role of SFAs for inflammatory response via TLR4-NF-kappaB signaling in vascular cells. Moreover, we propose that SPC can be useful as a selective inhibitor to suppress the TLR4-mediated inflammatory signaling.

Protein-tyrosine phosphatase 1B (PTP1B) is a major regulator of insulin sensitivity. We have described a novel action of PTP1B in the induction of sterol regulatory element-binding protein-1 (SREBP-1) gene expression through activation of protein phosphatase 2A (PP2A). PTP1B is anchored to the endoplasmic reticulum membrane via its C-terminal tail. We have previously reported that membrane localization of PTP1B is essential for PP2A activation, which is crucial for enhancing SREBP-1 gene expression in in vitro experiments. In this study, we further investigated the physiological importance of membrane localization of PTP1B in vivo. We found that transient liver-specific overexpression of wild-type PTP1B (PTP1B-WT) using adenovirus-mediated gene transfer was associated with hypertriglyceridaemia and enhanced hepatic SREBP-1 gene expression in mice. However, overexpression of the C-terminal truncated PTP1B (PTP1BDeltaCT) failed to increase hepatic SREBP-1 expression or serum triglyceride levels, despite causing insulin resistance. Our results indicate that activation of PTP1B in the liver could induce hypertriglyceridaemia and that anchoring of PTP1B to the membrane is crucial for its action.

Background Higher arterial stiffness and greater peripheral vascular resistance reduce blood flow in lower-leg arteries and contribute to the development of ischaemic limb in diabetic patients even without peripheral artery occlusive disease. The aim of this study was to clarify whether these vascular parameters are associated with long-term hyperglycaemia in diabetic patients. Methods We examined 45 type 2 diabetic patients and 38 age-matched nondiabetic subjects without peripheral artery occlusive disease assessed by ankle-brachial index consecutively admitted to our hospital, and followed them over a 3-year period (3.7 ± 0.7 years) with no vasodilative medication. Blood flow and resistive index, a measure of peripheral vascular resistance, at the popliteal artery were evaluated using gated two-dimensional cine-mode phase-contrast magnetic resonance imaging. Brachial-ankle pulse wave velocity was measured to assess arterial stiffness. Results At baseline, consistent with our previous report, diabetic patients showed higher brachial-ankle pulse wave velocity (p < 0.0001) and resistive index (p < 0.0001) and lower flow volume (p = 0.0044) than those of nondiabetic subjects. Stepwise multiple regression analysis revealed that duration of diabetes, mean HbA1c during the study, use of renin-angiotensin system inhibitors and change per year in resistive index were identified as significant independent variables predicting change per year in blood flow (r2 = 0.733, p < 0.0001) in diabetic patients. Mean HbA1c during the study was positively correlated with changes per year in brachial-ankle pulse wave velocity (p = 0.00007) and resistive index (p = 0.0014) and was negatively correlated with that in blood flow (p < 0.0001) in diabetic patients. Conclusions Long-term hyperglycaemia is a major cause of impaired peripheral circulation in lower-leg arteries in diabetic patients without peripheral artery occlusive disease. Copyright © 2009 John Wiley & Sons, Ltd.

We previously reported an association between the activating protein (AP)-2beta transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with a disease-susceptible allele of AP-2beta showed stronger AP-2beta expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta leads to lipid accumulation by enhancing glucose transport and inducing insulin resistance in 3T3-L1 adipocytes. In this study, we found that overexpression of AP-2beta in 3T3-L1 adipocytes accelerated the promoter activity of monocyte chemoattractant protein-1 (MCP-1) and subsequently increased both mRNA and protein expression and protein secretion. Furthermore, knockdown of endogenous AP-2beta by RNA interference reduced the mRNA and the protein expression of MCP-1. EMSAs and chromatin immunoprecipitation assays revealed specific binding of AP-2beta to MCP-1 promoter regions, in vitro and in vivo. Additionally, site-directed mutagenesis of the AP-2 binding site located at -137 to -129 relative to the transcription start site markedly diminished MCP-1 promoter activity, whereas other putative AP-2 binding sites did not. Our results clearly show that AP-2beta directly enhanced MCP-1 secretion by binding to its promoter. Thus, we propose that AP-2beta positively regulates MCP-1 expression; subsequently contributes to the infiltration of macrophages to adipose tissue; and leads to insulin resistance, type 2 diabetes, and cardiovascular diseases.

Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor in stimulating lipogenesis in the liver. Protein-tyrosine phosphatase 1B (PTP1B) induces SREBP-1 gene expression via protein phosphatase 2A (PP2A) activation. PTP1B is reported to be anchored on the endoplasmic reticulum (ER) via its C-terminal tail, and change in intracellular localization of PTP1B by C-terminal-truncation did not alter its inhibitory effects on insulin signaling. In this study, we investigated whether the change in intracellular localization of PTP1B could influence SREBP-1 gene expression. Overexpression of C-terminal truncated PTP1B (PTP1BdeltaCT) in rat Fao cells did not induce SREBP-1 gene expression. Furthermore, PTP1BdeltaCT failed to bind PP2A, resulting in impaired PP2A activation, whereas overexpression of wild-type PTP1B (PTP1BWT) associated with PP2A. Moreover, a membrane-targeted PTP1BDeltaCT activated PP2A with restored PP2A binding, despite the absence of its C-terminal region. Finally, overexpression of PTP1BdeltaCT into mouse primary cultured hepatocytes failed to enhance SREBP-1c mRNA, whereas membrane-targeted PTP1BdeltaCT led to enhanced SREBP-1c mRNA in hepatocytes as well as PTP1BWT. In conclusion, membrane localization of PTP1B is essential for PP2A activation, which is crucial for its enhancement of SREBP-1 gene expression.

Endothelial dysfunction is an early marker of atherosclerosis. Pioglitazone is commonly used in the treatment of type 2 diabetes and has vascular protective effects beyond its hypoglycemic ones. We investigated the vascular effects of short-term, low-dosage pioglitazone in patients with type 2 diabetes. The study included 15 subjects with type 2 diabetes with normoalbuminuria (age, 60.7 +/- 11.9 years; body mass index [BMI], 23.9 +/- 3.3 kg/m2). The patients received pioglitazone at 15 mg daily for 4 weeks. BMI, systolic and diastolic blood pressure, laboratory parameters (fasting plasma glucose, insulin, lipid profile, high-sensitive C-reactive protein [hsCRP], and adiponectin) were assessed at baseline and after treatment. The forearm blood flow (FBF) was measured during reactive hyperemia by strain-gauge plethysmography. Short-term, low-dosage pioglitazone did not improve glycemic control or insulin sensitivity. However, the peak FBF and flow debt repayment (FDR) were markedly improved. There was no correlation of the improvement of peak FBF and FDR with the observed changes of metabolic parameters. However, the increment of adiponectin and decrement of hsCRP were well correlated with the improvement of peak FBF. These results indicate that short-term low-dosage pioglitazone may improve vascular function via increasing adiponectin expression and decreasing low-grade inflammation in type 2 diabetic patients.

Prostate cancer (PCa) preferentially metastasizes to bone resulting in osteoblastic lesions with underlying osteolytic activities. The mechanisms through which PCa cells promote osteolytic activities and subsequent osteoblastic bone formation remain poorly understood. Parathyroid hormone-related protein (PTHrP), produced by bone cells and PCa, binds to receptors on osteoblasts and stimulates bone formation and resorption. We have previously reported that MCP-1 acts as a paracrine and autocrine factor for PCa progression. However, the role of PTHrP in regulating MCP-1 expression in bone microenvironment, specifically by human bone marrow endothelial cells (HBME) and osteoblasts (hFOB), as well as by PCa cells, has not been studied. Accordingly, we first determined the effect of PTHrP on MCP-1 expression by bone cells and PCa cells. PTHrP induced both MCP-1 protein and mRNA expression by HBME and hFOB cells, but not by PCa LNCaP and PC3 cells. To further determine the mechanisms of PTHrP-induced MCP-1 transcription, analysis of the MCP-1 promoter was performed. MCP-1 promoter activity was induced by PTHrP. Both C/EBPbeta and NF-kappaB binding elements are required for PTHrP-induced MCP-1 transcription. Finally, when a constitutively-active PTH receptor construct was transfected into HBME and hFOB cells, MCP-1 production was increased. The conditioned media collected from these cells induced osteoclast differentiation and PC3 proliferation and invasion in vitro. These inductions were partially inhibited by MCP-1 neutralizing antibody. We conclude that PTHrP-induced MCP-1 production by HBME and hFOB cells promotes osteoclast differentiation in vitro and such induction may play a critical role in PCa development in the bone microenvironment.

Recent studies have suggested that basic leucine zipper transcription factor MafA has a crucial role in pancreatic β-cell-specific insulin gene transcription. Thus, we investigated whether MafA overexpression in the intestine induces insulin production in small-intestinal epithelial cells in vivo. Recombinant adenovirus containing MafA gene (Ad-MafA) was prepared and administered orally to streptozocin-treated diabetic rats. Insulin gene expression was observed in the intestine by RT-PCR analysis, and then insulin protein was detected by immunohistochemical analysis after Ad-MafA administration. Furthermore, MafA overexpression in the intestine increased plasma insulin levels and ameliorated hyperglycemia. These results indicate that MafA overexpression in the intestine induces intestinal epithelial cells newly to produce and release insulin.

We previously reported the association between the activating enhancer-binding protein-2beta (AP-2beta) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with the disease-susceptible allele of AP-2beta showed stronger expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta leads to lipid accumulation by enhancing glucose transport and inducing insulin resistance in 3T3-L1 adipocytes. In this study we demonstrated that overexpression of AP-2beta in 3T3-L1 adipocytes decreased the expression and secretion of adiponectin and increased those of interleukin-6 (IL-6). Interestingly, the effects of AP-2beta on the expressions of adiponectin and IL-6 and the mechanisms by which AP-2beta modulated their expressions were different. We found that the promoter activity of adiponectin gene was inhibited by AP-2beta overexpression and enhanced by knockdown of endogenous AP-2beta, whereas IL-6 was unaffected. Electrophoretic mobility shift assays revealed the existence of putative responsive elements for AP-2beta and NF-YA in human and mouse adiponectin promoter regions, and mutation of this AP-2beta binding site abolished the inhibitory effect of AP-2beta. Furthermore, chromatin immunoprecipitation assays demonstrated that AP-2beta and NF-YA competitively bind to the same region of the adiponectin promoter. Our results clearly demonstrated that AP-2beta directly inhibits adiponectin gene expression by displacing NF-YA and binding to its promoter. We conclude that AP-2beta might modulate the expression of adiponectin by directly inhibiting its transcriptional activity.

Objective To assess whether an association between sodium-sensitive hypertension and metabolic syndrome exists; and whether, in patients with metabolic syndrome, the nocturnal fall of blood pressure decreases and salt restriction affects the circadian blood pressure rhythm. Methods Japanese patients with essential hypertension, who were treated without any antihypertensive agent, were maintained on a high-sodium diet and a low-sodium diet for 1 week each. On the last day of each diet, the 24-h blood pressures were measured. A diagnosis of metabolic syndrome was made according to the International Diabetes Foundation definition Results Among the 56 patients with essential hypertension, 15 patients were complicated with metabolic syndrome while 41 patients were not. The nocturnal blood pressure fall was significant in patients without metabolic syndrome, while it was not so in patients with metabolic syndrome. Only in patients with metabolic syndrome was the nocturnal blood pressure fall enhanced by sodium restriction. The prevalence of sodium-sensitive hypertension in patients with metabolic syndrome was significantly higher than in those without metabolic syndrome (70.6 versus 36.0%, respectively; P = 0.017). A multiple logistic regression analysis revealed central obesity to be an independent risk factor for sodium-sensitive hypertension (odds ratio, 1.41; 95% confidence interval, 1.04-1.91). Conclusions In patients with essential hypertension, an inter-relationship exists among metabolic syndrome, enhanced sodium sensitivity of the blood pressure and non-dipping. The elevated risk of cardiovascular diseases in patients with metabolic syndrome may be related to sodium-sensitive hypertension and non-dipping.

To compare F-18-fluorodeoxyglucose (FDG) and F-18-fluorothymidine (FLT) PET/CT examinations for differentiating between benign and malignant adrenal tumours.Thirty lipid-poor benign and 11 malignant tumours of 40 patients were included. FDG- and FLT-based indices including visual score, maximum standardized uptake value (SUVmax) and FDG adrenal lesion/liver SUVmax (A/L SUVmax) or FLT adrenal lesion/back muscle SUVmax (A/B SUVmax) ratio were compared between benign and malignant tumours using the Mann-Whitney's U or Wilcoxon signed-rank test, and their diagnostic performances were evaluated by means of the area under the curve (AUC) values derived from the receiver operating characteristic analysis.All indices were significantly higher in malignant than benign tumours on both images (p 0.05 each). On FDG-PET/CT, the sensitivity, specificity, and accuracy were 91 %, 63 % and 71 % for visual score, 91 %, 67 % and 73 % for SUVmax, and 100 %, 70 % and 78 % for A/L SUVmax ratio, respectively. On FLT-PET/CT, they were 100 %, 97 % and 98 % for visual score, SUVmax and A/B SUVmax ratio, respectively. All FLT indices were significantly higher than those of FDG in AUC (p 0.05 each).FLT-PET/CT may be superior to FDG-PET/CT in differentiating lipid-poor benign from malignant adrenal tumours because of higher specificity and accuracy.• All FDG indices were significantly higher in malignant than in benign tumours. • All FLT indices were significantly higher in malignant than in benign tumours. • All FLT indices were significantly higher than those of FDG in AUC.

OBJECTIVE—To clarify whether stiffness and impaired blood flow in lower-leg arteries are associated with severity of coronary artery calcification among asymptomatic diabetic patients. RESEARCH DESIGN AND METHODS—We enrolled 102 asymptomatic type 2 diabetic patients with no history of cardiovascular complications consecutively admitted to our hospital. Agatston coronary artery calcium (CAC) score, as a marker of coronary artery calcification, was obtained using electron-beam computed tomography. Total flow volume and resistive index, as an index of vascular resistance, at the popliteal artery were evaluated using gated two-dimensional cine-mode phase-contrast magnetic resonance imaging. Brachial-ankle pulse-wave velocity (PWV), as an index of distensibility in the lower-extremity arteries, was also measured using an automatic device. RESULTS—When the patients were grouped according to CAC scores of 0–10 (n = 54), 11–100 (n = 25), and >100 (n = 23), those with the highest scores, which is considered to show possible coronary artery disease, showed the highest brachial-ankle PWV (P < 0.001) and resistive index (P < 0.001) and the lowest total flow volume (P < 0.001) among the groups. Simple linear regression analyses showed that both brachial-ankle PWV (r = 0.508, P < 0.001) and resistive index (r = 0.500, P < 0.001) were positively correlated and total flow volume (r = −0.528, P < 0.001) was negatively correlated with the log-transformed CAC score. Receiver operator characteristic curve analyses indicated that 1,800 cm/s for brachial-ankle PWV, 1.03 for resistive index, and 70 ml/min for total flow volume were diagnostic values for identifying patients with the highest scores. CONCLUSIONS—Quantitatively assessed stiffness and impaired blood flow in lower-leg arteries may help identify diabetic patients with possible coronary artery disease.

Since World War II (WWII), exposures to westernized lifestyle have occurred in many non-Western countries, including Japan. The man born after WWII in Japan have had major changes in diet and other lifestyle factors, including heavy cigarette smoking, increase in the levels of total and low density lipoprotein cholesterol (LDL-C), increase in cholesterol intake in diet which is higher than in the United States (US), increase in body weight and obesity, substantial increase in alcohol consumption, and increase in western foods 1. Comparing the survey data from national representative samples around 1990, risk factor profiles for coronary heart disease (CHD): total cholesterol, and systolic and diastolic blood pressure, were very similar in men in the post WWII birth cohorts in the US and Japan, except for much higher prevalence of cigarette smoking in Japan and much higher prevalence of obesity in the US 2. We compared CHD mortality in men aged 35-44 between the US and Japan and found that CHD mortality in Japan is still considerably lower than in the US: 23.7/100,000 for the US and 8.7/100,000 for Japan 3. After careful review, the differences do not appear to be explained by differences in potential misclassification of causes of death 3. These observations imply that there may be important protective factors that reduce risk of CHD in Japan. Differences in genetic factors are unlikely to fully explain the difference in CHD mortality because studies of Japanese migrants to the US clearly illustrated the increase in CHD morbidity and mortality in Japanese men in the US 4. Epidemiological investigations monitoring and comparing CHD morbidity and mortality in the post WWII birth cohorts, i.e. age

During differentiation, expression of protein phosphatase-2Calpha (PP2Calpha) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Calpha in insulin signaling, we overexpressed wild-type (WT) PP2Calpha by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Calpha-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Calpha-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Calpha(R174G) did not produce such effects. Furthermore, overexpression of PP2Calpha-WT (but not PP2Calpha(R174G)) decreased the (32)P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Calpha activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Calpha by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Calpha antibody or PP2Calpha small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Calpha is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.

Pancreatic duodenal homeobox-1 (Pdx1) is a transcription factor, and its phosphorylation is thought to be essential for activation of insulin gene expression. This phosphorylation is related to a concomitant shift in molecular mass from 31 to 46 kDa. However, we found that Pdx1 was modified by SUMO-1 (small ubiquitin-related modifier 1) in β-TC-6 and COS-7 cells, which were transfected with Pdx1 cDNA. This modification contributed to the increase in molecular mass of Pdx1 from 31 to 46 kDa. Additionally, sumoylated Pdx1 localized in the nucleus. The reduction of SUMO-1 protein by use of RNA interference (SUMO-iRNAs) resulted in a significant decrease in Pdx1 protein in the nucleus. A 34-kDa form of Pdx1 was detected by the cells exposed to SUMO-iRNAs in the presence of lactacystin, a proteasome inhibitor. Furthermore, the reduced nuclear sumoylated Pdx1 content was associated with significant lower transcriptional activity of the insulin gene. These findings indicate that SUMO-1 modification is associated with both the localization and stability of Pdx1 as well as its effect on insulin gene activation.

To quantitatively evaluate the efficacy of methylprednisolone pulse therapy for extraocular muscle inflammation in thyroid-associated ophthalmopathy (TAO) using the short-tau inversion-recovery (STIR) technique of magnetic resonance imaging (MRI). The signal intensities of the superior rectus (SR), inferior rectus (IR), lateral rectus (LR), medial rectus (MR), and superior oblique (SO) muscles were measured using the STIR images from 34 eyes of 17 patients with TAO before and after methylprednisolone pulse therapy and 19 eyes of 19 controls. The signal intensity ratio (SIR) of the signal intensity in muscles to that in brain white matter was calculated. The mean SIRs of the controls were 1.08 ± 0.26 in the SR, 1.32 ± 0.29 in the IR, 1.34 ± 0.19 in the LR, 1.47 ± 0.25 in the MR, and 1.28 ± 0.22 in the SO muscles. SIRs exceeding 2.0 were out of the normal range. The SIRs of the patients with TAO before treatment were 2.19 ± 0.64, 2.44 ± 0.58, 1.96 ± 0.43, 2.24 ± 0.47, and 1.91 ± 0.42, respectively, which was significantly (P

The term "diabetic neuropathy" refers to many varieties of neuropathies, including diabetic peripheral neuropathies (DPNs). DPNs are categorized into generalized and focal/multifocal varieties. Diabetic sensorimotor polyneuropathy (DSPN) and diabetic autonomic neuropathy (DAN) are typical DPNs, and their development is clearly linked to hyperglycemia and subsequent metabolic and ischemic change. On the other hand, other forms of neuropathy, including multifocal diabetic neuropathies (e.g., lumbosacral, thoracic, and cervical radiculoplexus neuropathies) are thought to be associated with inflammatory or immune processes. Diabetic patients can also develop chronic inflammatory demyelinating polyneuropathy (CIDP). CIDP in diabetic patients (DM-CIDP) should be ruled out, especially in patients with advanced DSPN. Recently, it was reported that diabetic radiculoplexus neuropathies as well as CIDP respond favorably to immunotherapy. Thus, these immune-mediated diabetic neuropathies are treatable, and should be differentiated from advanced DSPN.

OBJECTIVES We investigated whether abnormal pteridine metabolism is related to coronary endothelial dysfunction in insulin-resistant subjects. BACKGROUND Depletion of tetrahydrobiopterin (BH 4 ) and elevation of the 7,8-dihydrobiopterin (BH 2 ) (activating and inactivating cofactors of nitric oxide synthase [NOS], respectively) contribute to impairment of NO-dependent vasodilation through reduction of NOS activity as well as increased superoxide anion generation in insulin-resistant rats. METHODS Thirty-six consecutive nondiabetic, normotensive and nonobese subjects with angiographically normal coronary vessels were studied. Traditional coronary risk factors, plasma pteridine levels, activities of erythrocyte dihydropteridine reductase (DHPR), the recycling enzyme that converts BH 2 to BH 4 and lipid peroxide (LPO) levels were measured and coronary endothelial function was assessed with graded infusions of acetylcholine (ACh). RESULTS When we divided patients into tertiles based on insulin sensitivity, we observed stepwise decreases in the maximal ACh-induced vasodilation and plasma BH 4 /7,8-BH 2 ratio, and increases in coronary LPO production as insulin sensitivity decreased. The ACh-induced vasodilation was positively correlated with insulin sensitivity, BH 4 /7,8-BH 2 ratio and DHPR activity. Furthermore, BH 4 /7,8-BH 2 was inversely correlated with DHPR activity and insulin sensitivity. In multiple stepwise regression analysis, BH 4 /BH 2 was independently related to ACh-induced vasodilation and accounted for 39% of the variance. However, no significant correlation existed between other traditional risk factors and BH 4 /7,8-BH 2 . CONCLUSIONS These results indicate that both abnormal pteridine metabolism and vascular oxidative stress are linked to coronary endothelial dysfunction in the insulin-resistant subjects.

Abstract —We have reported that a deficiency of tetrahydrobiopterin (BH 4 ), an active cofactor of endothelial NO synthase (eNOS), contributes to the endothelial dysfunction through reduced eNOS activity and increased superoxide anion (O 2 − ) generation in the insulin-resistant state. To further confirm this hypothesis, we investigated the effects of dietary treatment with BH 4 on endothelium-dependent arterial relaxation and vascular oxidative stress in the aortas of insulin-resistant rats. Oral supplementation of BH 4 (10 mg · kg −1 · d −1 ) for 8 weeks significantly increased the BH 4 content in cardiovascular tissues of rats fed high levels of fructose (fructose-fed rats). Impairment of endothelium-dependent arterial relaxation in the aortic strips of the fructose-fed rats was reversed with BH 4 treatment. The BH 4 treatment was associated with a 2-fold increase in eNOS activity as well as a 70% reduction in endothelial O 2 − production compared with those in fructose-fed rats. The BH 4 treatment also partially improved the insulin sensitivity and blood pressure, as well as the serum triglyceride concentration, in the fructose-fed rats. Moreover, BH 4 treatment of the fructose-fed rats markedly reduced the lipid peroxide content of both aortic and cardiac tissues and inhibited the activation of 2 redox-sensitive transcription factors, nuclear factor-κB and activating protein-1, which were increased in fructose-fed rats. The BH 4 treatment of control rats did not have any significant effects on these parameters. These results indicate that BH 4 augmentation is essential for the restoration of eNOS function and the reduction of vascular oxidative stress in insulin-resistant rats.

Aims/hypothesis. We studied 76 patients with Type II (non-insulin-dependent) diabetes mellitus and 16 age-matched non-diabetic subjects (control group) to clarify qualitative and quantitative abnormalities of waveform and flow volume of the popliteal artery. Methods. The 76 diabetic patients comprised 16 patients with occlusive arterial disease in the lower extremities [arteriosclerosis obliterans (ASO) group] and 60 patients free from this disease (non-ASO group). We flow analysed the popliteal artery and measured the phosphocreatine to inorganic phosphate ratio of resting plantar muscles to identify risk factors for foot lesions using gated magnetic resonance two-dimensional cine-mode phase-contrast imaging and 31P spectroscopy. Results. The control and non-ASO groups had a triphasic waveform with systolic, early and late diastolic components. All ASO patients had an abnormal monophasic waveform and a lower ankle brachial index than that of the control and non-ASO groups. To clarify the mechanism of reduced flow volume of lower extremities, we assigned the 60 patients of the non-ASO group to the three subgroups based on their levels of total flow volume of the popliteal artery. The lowest group showed an abnormal triphasic waveform with lower amplitudes of systolic and late diastolic components and flow velocities in foot arteries than those of the highest group although ABI was similar. From stepwise multiple regression analysis, late diastolic flow volume was identified as an independent determinant for the phosphocreatine to inorganic phosphate ratio (r 2 = 0.484, p < 0.001). Conclusion/interpretation. Waveform analysis of popliteal artery provides a powerful tool for identifying impaired peripheral circulation caused by either occlusive arterial disease or increased arterial resistance in diabetic patients. [Diabetologia (2000) 43: 1031–1038]

Previously, we have reported that the lipoprotein fraction containing intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) isolated from diabetics stimulates an atherogenic cytokine in cultured endothelial cells. To study which lipoprotein fraction isolated from diabetics can modulate the gene expression in endothelial cells, we isolated IDL and LDL fractions from 14 type 2 diabetics and seven age- and BMI- adjusted non-diabetics. We measured the effects of the lipoproteins on mRNA expression of atherogenic molecules in cultured endothelial cells. We found that the IDL fraction stimulated monocyte chemoattractant protein-1 (MCP-1) mRNA expression in endothelial cells as time- and dose-dependent fashions, while the LDL fraction was not effective. IDL isolated from diabetics also increased not only platelet-derived growth factor B-chain, but also intercellular adhesion molecule-1 mRNA contents. Furthermore, the HbA(1c) levels in diabetics were significantly correlated with their abilities of IDL to increase MCP-1 mRNA content in the cells and the increment coincided with the increase in MCP-1 protein release into culture media. These results indicate that qualitative as well as quantitative changes in IDL fraction in diabetes are atherogenic through stimulating gene expression of atherogenic molecules in endothelial cells.

Aims/hypothesis. We studied 36 Type II (non-insulin-dependent) diabetic patients without occlusive arterial diseases in the lower extremities and 12 age-matched and sex-matched non-diabetic subjects to clarify the association between diabetic polyneuropathy and foot ulcers using 1H- and 31P-magnetic resonance spectroscopy and imaging.¶Methods. The 36 diabetic patients consisted of 12 patients with superficial foot ulcers and 24 patients free from this disease. We measured fat to water and phosphocreatine to inorganic phosphate (PCr:Pi) ratios and calculated the intracellular pH of resting plantar muscles by depth-resolved surface-coil spectroscopy using an 1H-31P double tuned coil. Furthermore, foot vasculature, fat and PCr contents of plantar muscles were visualised by phase-contrast angiography, T1-weighted spin-echo imaging and 31P-chemical shift imaging.¶Results. The 12 foot ulcer patients showed a reduced PCr to Pi ratio (p < 0.001) and peripheral nerve functions (p < 0.01–0.001) but an increased fat to water ratio (p < 0.001) and intracellular pH (p < 0.001) compared with the 24 patients without ulcers. From stepwise multiple regression analyses, motor nerve function as well as severity of nephropathy was associated with both fat to water and PCr to Pi ratios. When these patients were categorised into three groups based on their level of motor nerve function, the frequency of foot ulcers of the lowest group was higher than that of the highest group.¶Conclusion/interpretation. Our findings indicated that motor nerve dysfunction in diabetic patients was closely associated with impaired energy metabolism, fatty infiltration and increased intracellular pH of plantar muscles and high frequency of foot ulcers. These new techniques could contribute to help clarify the predisposing factors for foot ulcers. [Diabetologia (2000) 43: 165–172]

Objective Evaluation of metabolic states and chronic complications is essential for maintaining a high quality of care for diabetic patients. We have assessed the quality of care in routine outpatient clinics for diabetic subjects in our university hospital, and compared with those in a newly introduced standardized clinic to evaluate the new care system. Methods The quality of care was assessed by the chart review in 1995, and compared with those from 1996-1997 in the "Diabetes Follow-up Clinic" which is systematically designed for the standardized care. Patients The subjects were recruited among 860 patients who visited the outpatient clinic in July and August of 1995 with a diagnosis of diabetes or glucose intolerance. Six hundred seventy-two patients whose follow-up period had been more than 6 months with clinically diagnosed diabetes were used for the analysis. Results Laboratory tests such as determination of HbA1c, and serum levels of lipids and creatinine were performed in more than 90% of the patients in the routine outpatient clinics. However, ophthalmology referral, 24-hour urine collection for the determination of creatinine clearance and albumin excretion, and electrocardiograms were not well performed and were incompletely documented (40-60% of the patients within a previous year and 70-80% in the last 2 years). In the standardized "Diabetes Follow-up Clinic", only four out of 555 diabetic patients failed to collect their 24-hour urine, and all participants had ankle blood pressure measurements, nerve conduction study, and nylon monofilament tests, etc. Furthermore, more than 95% of the patients had funduscopic examinations by ophthalmologists as well as records of electrocardiogram. Conclusion Introduction of the standardized "Diabetes Follow-up Clinic" may be one of the choices for increasing the quality of outpatient care and for the prevention of chronic diabetic complications.(Internal Medicine 39: 783-787, 2000)

A newly identified 3'-untranslated region (UTR) polymorphism of the gene for skeletal muscle-specific glycogen-targeting subunit of protein phosphatase 1 (PPP1R3) was associated with insulin resistance and type 2 diabetes in Pima Indians (Xia J, Scherers W, Cohen PTW, Majer M, Xi T, Norman RA, Knowler WC, Bogardus C, Prochazka M: A common variant in PP1R3 associated with insulin resistance and type 2 diabetes. Diabetes 47:1519-1524, 1998). Thus, we investigated the frequency of polymorphism of the adenine- and thymine-rich element (ARE-1 and its variant ARE-2) in 426 Japanese type 2 diabetic and 380 nondiabetic subjects using a polymerase chain reaction (PCR)-restriction enzyme fragment length polymorphism (RFLP) method. The allele frequency of the ARE-2 variant in diabetic subjects was higher than that in nondiabetic subjects (0.34 vs. 0.29; P < 0.05), even though its frequency in Japanese subjects was lower (P < 0.001) than the reported value in Pima Indians (0.56). An aspartate polymorphism at codon 905 was 100% coupled to the ARE-2 allele, and its allele frequency was higher also in diabetic subjects. Although a serine substitution at codon 883 was partially linked with the ARE-2 allele, there was no difference between diabetic and nondiabetic subjects. These results indicate that the frequency of polymorphism of the PPP1R3 gene (ARE-2 and Asp905) is different between two ethnic groups and is increased in Japanese people with type 2 diabetes, suggesting that these variants may be a possible marker for searching for diabetogenic genes.

To investigate the normalization of enhanced oxidative modification of the lipoprotein such as increased lysophosphatidylcholine (LPC) and lipid hydroperoxide (LPO) contents in diabetic subjects, we studied the effect of cholesterol-lowering treatment on those parameters in 24 hypercholesterolemic Type 2 diabetic patients. Those patients were randomly assigned to two treatment groups, such as 12 patients treated with pravastatin 10 mg daily and 12 patients treated with probucol 500 mg daily for 8 weeks. Characteristics of the patients including age, gender, body mass index (BMI), smoking habit, modality of diabetic treatment and the glycemic control state were comparable between the two groups. LPC content in the lipoprotein fractions obtained from 24 patients with Type 2 diabetes mellitus was significantly higher than that of non-diabetic control subjects. The abnormality was improved to the control level after a significant improvement of serum cholesterol levels following 8 week-treatments with either probucol or pravastatin without any change in glycemic control (P < 0.025). Furthermore, increased LPO content in the lipoprotein fraction in those diabetics was also significantly (P < 0.0025) improved by the probucol treatment and tended to be improved by pravastatin treatment (P = 0.06). LPC contents in the lipoprotein fraction was positively correlated with LPO contents before cholesterol-lowering treatments (r = 0.41, P < 0.05). These results indicate that cholesterol-lowering treatments effectively reduce oxidative modification of the lipoprotein fraction containing intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) in hypercholesterolemic Type 2 diabetic patients.