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Background: This study was aimed to compare the efficacy of two combination tablets of dipeptidyl peptidase-4 (DPP-4) inhibitors and metformin with different dosages, alogliptin/metformin (AM) and vildagliptin/metformin (VM), on glycemic control in patients with type 2 diabetes (T2D). Methods: This was a prospective, multicenter, open-label, randomized, parallel group, comparative trial. After a run-in period of treatment with metformin alone, a total of 59 Japanese outpatients with T2D, aged 20–79 years with glycated hemoglobin (HbA1c) levels of 6.5%–10% were randomly assigned to 12-week AM treatment, alogliptin 25 mg/metformin 500 mg combination tablet orally once a day, or VM treatment, vildagliptin 50 mg/metformin 250 mg combination tablet orally twice a day. The primary endpoints were the changes in HbA1c and fasting plasma glucose (FPG) levels from baseline to week 12 between the two groups. Blinded intermittently scanned continuous glucose monitoring (isCGM) was performed between weeks 10 and 12. The incidence of adverse events during the study was also evaluated. Results: In all, 52 participants were analyzed. Significant decreases in HbA1c and FPG levels from baseline to week 12 were observed in both treatment groups. However, there were no significant differences between the AM and VM groups in the change in HbA1c level (–0.3% and –0.4%, P = 0.309) or the FPG level (–9.0 and –15.0 mg/dL, P = 0.789). The isCGM revealed that both treatments achieved the recommended glycemic target range. No adverse events, such as severe hypoglycemia, were observed in either group. Conclusions: We concluded that there were no significant differences in the efficacy of two combination tablets of DPP-4 inhibitors and metformin with different dosages on glycemic control in patients with T2D. [ABSTRACT FROM AUTHOR]

The diagnosis of insulinoma in perinatal women can be difficult, as hypoglycemic symptoms may be masked by pregnancy‐associated insulin resistance. In addition, when multiple insulinomas are observed, it is necessary to consider the possibility not only of MEN1, but also of insulinomatosis. [ABSTRACT FROM AUTHOR]

Objective Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) possess multiple bioactive isoforms that are rendered non-insulinotropic by the enzyme dipeptidyl peptidase-4 (DPP-4). Recently, some ELISA kits have been developed to specifically measure “active” GIP and GLP-1, but it is unclear if these kits can accurately quantify all bioactive forms. Therefore, it remains uncertain to what extent treatment with a DPP-4 inhibitor boosts levels of biologically active GIP and GLP-1. Thus, we evaluated our novel receptor-mediated incretin bioassays in comparison to commercially available ELISA kits using plasma samples from healthy subjects before and after DPP-4 inhibitor administration. Methods We utilized cell lines stably co-transfected with human GIP or GLP-1 receptors and a cAMP-inducible luciferase expression construct for the bioassays and commercially available ELISA kits. Assays were tested with synthetic GIP and GLP-1 receptor agonists and plasma samples collected from subjects during a 75 g oral glucose tolerance test (OGTT) performed before or following 3-day administration of a DPP-4 inhibitor. Results A GIP isoform GIP(1–30) NH2 increased luciferase activity similarly to GIP(1–42) in the GIP bioassay but was not detectable by either a total or active GIP ELISA kit. During an OGTT, total GIP levels measured by ELISA rapidly increased from 0 min to 15 min, subsequently reaching a peak of 59.2 ± 8.3 pmol/l at 120 min. In contrast, active GIP levels measured by the bioassay peaked at 15 min (43.4 ± 6.4 pmol/l) and then progressively diminished at all subsequent time points. Strikingly, at 15 min, active GIP levels as determined by the bioassay reached levels approximately 20-fold higher after the DPP-4 inhibitor treatment, while total and active GIP levels determined by ELISA were increased just 1.5 and 2.1-fold, respectively. In the absence of DPP-4 inhibition, total GLP-1 levels measured by ELISA gradually increased up to 90 min, reaching 23.5 ± 2.4 pmol/l, and active GLP-1 levels determined by the bioassay did not show any apparent peak. Following administration of a DPP-4 inhibitor there was an observable peak of active GLP-1 levels as determined by the bioassay at 15 min after oral glucose load, reaching 11.0 ± 0.62 pmol/l, 1.4-fold greater than levels obtained without DPP-4 inhibitor treatment. In contrast, total GLP-1 levels determined by ELISA were decreased after DPP-4 inhibitor treatment. Conclusion Our results using bioassays indicate that there is a greater increase in plasma levels of bioactive GIP than GLP-1 in subjects treated with DPP-4 inhibitors, which may be unappreciated using conventional ELISAs. [ABSTRACT FROM AUTHOR]

A low carbohydrate diet (LCHD) as well as sodium glucose cotransporter 2 inhibitors (SGLT2i) may reduce glucose utilization and improve metabolic disorders. However, it is not clear how different or similar the effects of LCHD and SGLT2i are on metabolic parameters such as insulin sensitivity, fat accumulation, and especially gluconeogenesis in the kidney and the liver. We conducted an 8-week study using non-diabetic mice, which were fed ad-libitum with LCHD or a normal carbohydrate diet (NCHD) and treated with/without the SGLT-2 inhibitor, ipragliflozin. We compared metabolic parameters, gene expression for transcripts related to glucose and fat metabolism, and glycogen content in the kidney and the liver among the groups. SGLT2i but not LCHD improved glucose excursion after an oral glucose load compared to NCHD, although all groups presented comparable non-fasted glycemia. Both the LCHD and SGLT2i treatments increased calorie-intake, whereas only the LCHD increased body weight compared to the NCHD, epididimal fat mass and developed insulin resistance. Gene expression of certain gluconeogenic enzymes was simultaneously upregulated in the kidney of SGLT2i treated group, as well as in the liver of the LCHD treated group. The SGLT2i treated groups showed markedly lower glycogen content in the liver, but induced glycogen accumulation in the kidney. We conclude that LCHD induces deleterious metabolic changes in the non-diabetic mice. Our results suggest that SGLT2i induced gluconeogenesis mainly in the kidney, whereas for LCHD it was predominantly in the liver. [ABSTRACT FROM AUTHOR]

Tubular injury is one of the important determinants of progressive renal failure in diabetic nephropathy (DN), and TGF-β1 has been implicated in the pathogenesis of tubulointerstitial disease that characterizes proteinuric renal disease. The aim of this study was to identify novel therapeutic target molecules that play a role in the tubule damage of DN. We used an LC-MS/MS-based proteomic technique and human renal proximal epithelial cells (HRPTECs). Urine samples from Japanese patients with type 2 diabetes (n = 46) were used to quantify the candidate protein. Several proteins in HRPTECs in cultured media were observed to be driven by TGF-β1, one of which was 33-kDa IGFBP7, which is a member of IGFBP family. TGF-β1 up-regulated the expressions of IGFBP7 mRNA and protein in a dose- and time-dependent fashion via Smad2 and 4, but not MAPK pathways in HRPTECs. In addition, the knockdown of IGFBP7 restored the TGF-β1-induced epithelial to mesenchymal transition (EMT). In the immunohistochemical analysis, IGFBP7 was localized to the cytoplasm of tubular cells but not that of glomerular cells in diabetic kidney. Urinary IGFBP7 levels were significantly higher in the patients with macroalbuminuria and were correlated with age (r = 0.308, p = 0.037), eGFR (r = −0.376, p = 0.01), urinary β2-microglobulin (r = 0.385, p = 0.008), and urinary N-acetyl-beta-D-glucosaminidase (NAG) (r = 0.502, p = 0.000). A multivariate regression analysis identified urinary NAG and age as determinants associated with urinary IGFBP7 levels. In conclusion, our data suggest that TGF-β1 enhances IGFBP7 via Smad2/4 pathways, and that IGFBP7 might be involved in the TGF-β1-induced tubular injury in DN. [ABSTRACT FROM AUTHOR]

Persistent high concentration of glucose causes cellular stress and damage in diabetes via derangement of gene expressions. We previously reported high glucose activates hypoxia-inducible factor-1 α and downstream gene expression in mesangial cells, leading to an extracellular matrix expansion in the glomeruli. A glucose-responsive transcription factor carbohydrate response element-binding protein (Ch REBP) is a key mediator for such perturbation of gene regulation. To provide insight into glucose-mediated gene regulation in mesangial cells, we performed chromatin immunoprecipitation followed by DNA microarray analysis and identified platelet-derived growth factor-C ( PDGF-C) as a novel target gene of Ch REBP. In streptozotocin-induced diabetic mice, glomerular cells showed a significant increase in PDGF-C expression; the ratio of PDGF-C-positive cells to the total number glomerular cells demonstrated more than threefold increase when compared with control animals. In cultured human mesangial cells, high glucose enhanced expression of PDGF-C protein by 1.9-fold. Knock-down of Ch REBP abrogated this induction response. Upregulated PDGF-C contributed to the production of type IV and type VI collagen, possibly via an autocrine mechanism. Interestingly, urinary PDGF-C levels in diabetic model mice were significantly elevated in a fashion similar to urinary albumin. Taken together, we hypothesize that a high glucose-mediated induction of PDGF-C via Ch REBP in mesangial cells contributes to the development of glomerular mesangial expansion in diabetes, which may provide a platform for novel predictive and therapeutic strategies for diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Abstract: Aim: In order to clarify the importance of Interleukin (IL)-18 in the development of diabetic nephropathy (DN), we evaluated the expressions of IL-18 in diabetic kidney. Methods: We performed immmunohistochemical analysis of IL-18 and IL-18 receptor (IL-18 R) in human kidney tissue derived from 12 subjects with type 2 diabetes and overt nephropathy, and compared with those in 7 subjects with minimal change nephrotic syndrome (MCNS). In addition, we examined the regulation of IL-18 expression using human renal proximal tubular epithelial cells (HRPTECs) in culture. Results: IL-18 expression in tubular cells was observed in higher rate (83%) in patients with diabetes, whereas one positive specimen (14.3%) for IL-18 in patients with MCNS. In contrast, IL-18 R was expressed in glomerular mesangial cells and endothelial cells as well as tubular cells, similarly in almost of both groups. Exposure to transforming growth factor beta (TGF-β1) led to two-fold increase in IL-18 gene expression in HRPTECs, and mitogen-activated protein kinases (MAPK) inhibitors abolished TGF-β1-induced IL-18 mRNA expression. Western blot analysis showed the IL-18 protein in HRPTECs. Conclusion: The present data indicate that IL-18 is overexpressed in human tubular epithelial cells in diabetic nephropathy, probably through the activation of MAPK pathways induced by TGF-β1. [Copyright &y& Elsevier]

Renal damage in diabetic nephropathy (DN) is evaluated by biochemical tests such as urine albumin-to-creatinine ratio (UACR) and serum estimated glomerular filtration rate (eGFR). On the other hand, renal biopsy, which is rich in definitive information, is rarely performed because of its complexity and high invasion. To evaluate whether diffusion-weighted magnetic resonance imaging (DW-MRI) could be a noninvasive approach for distinguishing DN, we performed MRI scanning of the subjects with 6 b-values (b = 0, 50, 100, 350, 400, 700) and analyzed the parameters using the kidney imaging software (Hitachi, Ltd., Tokyo). A total of 40 subjects were enrolled, 25 of whom had type 2 diabetes with or without hypertension (T2D), 9 had nondiabetes but hypertension (HT), and 6 were healthy volunteers (Control). T2D showed lower values of apparent diffusion coefficient (ADC) 0-700 compared with HT (129.0 ± 2.12 x10-5mm2/s in T2D vs. 132.5 ± 1.43 ×10-5mm2/s in HT, p<0.001). In addition, T2D with HT (n=15) had lower ADC0-700 compared with T2D without HT (n=10) (p < 0.01). Interestingly, T2D with non-DN nor HT presented lower ADC0-700 compared with HT (p<0.05). ADC0-700 in T2D were correlated with serum eGFR (r = 0.40, p <0.05) and inversely correlated with serum creatinine (r = - 0.40, p <0.05), cystatin C (r = - 0.54, p <0.01) and UACR (r = - 0.57, p <0.01). On the other hand, ADC0-700 in HT were not correlated with any of parameters of the renal function. Multivariate regression analysis with a stepwise forward method identified UACR as the only factor associated with the ADC0-700 values (β = - 0.68, p <0.001). Accumulating evidences show that lower levels of ADC on DW-MRI indicate greater fibrosis in various organs. Therefore, cortical ADC0-700 could predict the progression of the renal damage such as tubulointerstitial fibrosis. DW-MRI might provide a convenient diagnostic tool in the evaluation of DN. Disclosure: H. Kitsunai: None. Y. Takiyama: Other Relationship; Self; Gilead Sciences, Inc., Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Mochida Pharmaceutical Co., Ltd., Roche Diabetes Care, Taisho Pharmaceutical Co., Ltd. N. Nakagawa: None. N. Hasebe: None. A. Okizaki: Research Support; Self; Fuji-Film Toyama Chemical Co., Ltd., Nihon Medhi-physics. M. Haneda: Advisory Panel; Self; Eli Lilly Japan K. K., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc. Funding: Japan Society for the Promotion of Science (20K17420) [ABSTRACT FROM AUTHOR]

Recent study has demonstrated the Nrf2 (NF-E2-related factor 2) activator bardoxolone methyl improved renal function in diabetic kidney disease (DKD). On the other hand, mitochondrial transcription factor A (TFAM) activation inhibits mitochondrial DNA (mtDNA) release into the cytoplasm and suppresses stimulator of interferon genes (STING)-mediated inflammation in renal proximal tubular cells. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is recognized as activators of Nrf2. In this study, we investigated the effects of SFN on TFAM and STING expression and its mitochondrial protective and anti-inflammatory effects. Because mitochondrial damage caused by fatty acids bound to albumin is involved in pathogenesis of DKD due to increased albumin reabsorption, we treated human renal proximal tubular cells (Lonza) with palmitic acid (PA, 200µM). Total RNA, DNA and protein were extracted and analyzed by qRT-PCR and western blotting. To quantify the mitochondrial DNA released into the cytoplasm, DNA was extracted from cytoplasmic and nuclear fractions. SFN (10µM) induced Nrf2 downstream target gene, NAD(P)H:quinone oxidoreductase 1 (NQO-1) mRNA expression to 359% (p<0.05). SFN alone increased TFAM mRNA expression to 132% (p<0.05) and suppressed STING mRNA expression by 26% (p<0.05). Whereas, PA had no effect on TFAM mRNA expression but increased STING mRNA expression to 135% (p<0.05). SFN suppressed PA-induced STING mRNA expression by 34% (p<0.05) and STING protein expression by 48%. PA increased cytoplasmic mtDNA to 237% and SFN decreased PA-induced cytoplasmic mtDNA release by 41%. PA increased IL-6 mRNA expression to 139% (p<0.05) and MCP-1 mRNA expression to 138% (p<0.05). SFN suppressed PA-induced IL-6 mRNA expression by 33% (p<0.05) and MCP-1 mRNA expression by 47% (p<0.05). Together, the Nrf2 activators may have renoprotective effects through mitochondrial protection and anti-inflammatory effect by activating TFAM and inhibiting STING. Disclosure: R. Bessho: None. T. Takiyama: None. S. Promsuwan: None. H. Kitsunai: None. K. Sawamoto: Other Relationship; Self; Abbott, Boehringer Ingelheim Pharmaceuticals, Inc., Merck & Co., Inc., Ono Pharmaceutical Co., Ltd., Roche Diagnostics K. K., Taisho Pharmaceutical Co., Ltd. Y. Takeda: None. Y. Takiyama: Other Relationship; Self; Gilead Sciences, Inc., Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Mochida Pharmaceutical Co., Ltd., Roche Diabetes Care, Taisho Pharmaceutical Co., Ltd. [ABSTRACT FROM AUTHOR]

We investigated whether overnutrition during pregnancy were associated with the changes in gene expressions, oxygen metabolism, and vascular and hepatic parenchymal remodeling in fetal livers at mid gestational days E14.5 of dams fed a control diet (CD group) or a high-fat diet (HFD group) during pregnancy. Synchrotron-based phase-contrast micro-CT demonstrated embryos of HFD group showed hepatomegaly accompanied with the same size umbilical vein and less portal branching, which might result in liver hypoxia. Correspondingly, fetal livers of HFD group increased the expression of HIF-1alpha. To identify genes and pathways targeted by maternal overnutrition in fetal livers, we assayed mRNA levels with microarray. We identified 430 differentially expressed genes (DEGs) including 146 upregulated- and 284 downregulated genes in fetal livers of HFD group compared with those of CD group. The up-regulated DEGs were significantly enriched in DNA alkylation, bile secretion, and the down-regulated DEGs mainly enriched in neuronal system, cell maturation. Among upregulated DEGs, a cancer/testis antigen HORMAD1 was induced by hypoxia up to 30-fold compared with that in normoxia in primary mouse hepatocytes (PMH), and the genetic inhibition of HIF-1alpha by siRNAs abolished the stimulatory effect of hypoxia on Hormad1 expression. In addition, we found three putative hypoxia response elements within the mouse Hormad1gene. Interestingly, the tumor suppressor WW domain-containing oxidoreductase (WWOX), which inhibits the activity of HIF-1alpha, were remarkably decreased in hypoxia in PMH. Finally, HORMAD1 protein was upregulated in fetal livers and NASH-based HCC in offspring of HFD group at 15 weeks old. Our results suggest that WWOX-HIF-1alpha-HORMAD1 pathway might be involved in the development of NAFLD in offspring of obese and/or diabetic pregnant woman, and be an important therapeutic target of NASH/HCC. Disclosure: T. Takiyama: None. Y. Takiyama: Other Relationship; Self; Gilead Sciences, Inc., Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Mochida Pharmaceutical Co., Ltd., Roche Diabetes Care, Taisho Pharmaceutical Co., Ltd. R. Bessho: None. H. Kitsunai: None. Y. Takeda: None. H. Sakagami: None. T. Sera: None. M. Nakamura: None. S. Horike: None. Y. Nishikawa: None. [ABSTRACT FROM AUTHOR]

Nonalcoholic steatohepatitis (NASH), a progressive form of nonalcoholic fatty liver disease (NAFLD), is associated with hepatocellular carcinoma (HCC). The worldwide prevalence of NAFLD in children suggests an early-life origin. To examine the impact of intrauterine nutrition on the development of NASH/HCC, female C57BL/6J mice were fed with either high-fat diet (HFD) or control diet (CD) during gestation. After weaning, the male offspring were fed with CD, generating two groups of male offspring, HFD and CD. HFD remarkably elevated plasma NEFA and ALT. Consistently, HFD had severe form of NASH, presenting the fibrosis, accompanied with HCC. Synchrotron micro-CT showed the architectural remodeling of the parenchyma in HFD at 14.5 days postcoitum (dpc), one week and 15 weeks of age, not in CD. In the liver of HFD at 14.5dpc, gene ontology term enrichment analysis suggested that up-regulated differentially expressed genes (DEGs) significantly enriched in DNA alkylation, bile secretion, and the down-regulated DEGs mainly enriched in neuronal system, cell maturation. KEGG pathway analysis found down-regulated DEGs significantly enriched in five pathways including Graft-versus-host disease (LogP -24.785) such as FasL, and up-regulated HIF-1 signaling pathway in the liver of HFD at one week of age. On the contrary, 15-week-old HFD had increased cytoplasmic pimonidazole staining, but no nuclear staining of HIF-1α in HCC cells, which suggests breakdown of hypoxic response mechanism, concomitantly with up-regulated miR199a-5p, miR-1949, miR214-3p and miR-3090-5p, which target HIF-1α. This study demonstrates that the early exposure to an overnutrition in utero, subsequent neonatal and adult nutritional alternation cause NASH/HCC in offspring, accompanied by multiple changes in gene expressions. This means a "multi-hit theory." Disclosure: T. Takiyama: None. R. Bessho: None. K. Sawamoto: None. Y. Takiyama: Other Relationship; Self; Abbott, Boehringer Ingelheim Pharmaceuticals, Inc., Merck & Co., Inc., Ono Pharmaceutical Co., Ltd., Roche Diagnostics K.K., Taisho Pharmaceutical Co., Ltd. [ABSTRACT FROM AUTHOR]

The renoprotective mechanism of SGLT2 inhibitors has not been fully elucidated. Tubular hypoxia is major driving force for proximal tubulopathy in diabetic kidney. In addition, diabetes causes dysregulation of AMP-activated protein kinase (AMPK) in kidney cortex. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on hypoxia inducible factor-1α (HIF-1α) expression and AMPK phosphorylation in cultured human renal proximal tubular epithelial cells (HRPTECs) and on tubulointerstitial pathological changes in diabetic mice. Luseogliflozin inhibited hypoxia (1%O2, 24h)-induced HIF-1α protein expression in HRPTECs in a dose-dependent manner (1-100µM). In addition, luseogliflozin increased AMPKα protein phosphorylation (Th172) in normoxic and hypoxic condition. Intriguingly, luseogliflozin suppressed oxygen consumption rate (OCR) measured by oxygen quenching phosphorescent probe by 69% in HRPTECs from normoxic condition. Hypoxia decreased OCR by 52% and luseogliflozin further decreased OCR by 33% from hypoxic condition. Moreover, luseogliflozin rescued hypoxic state in HRPTECs even under hypoxic conditions assessed by a hypoxia-sensitive dye, pimonidazole. These data indicate luseogliflozin inhibits HIF-1α expression through suppression of mitochondrial OCR, which leads to restore intracellular hypoxia. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin ameliorated tubular injury compared to non-treated db/db mice. Furthermore, immunostaining of HIF-1α and fibronectin in cortical tubules revealed that luseogliflozin decreased HIF-1α and fibronectin expression in db/db mice. In conclusion, luseogliflozin decreases mitochondrial OCR and ameliorates tubular fibrosis at least partly by suppressing HIF-1α expression and activating AMPK in renal proximal tubules of diabetic mice. These results may provide a novel renoprotective target of SGLT2 inhibitor. Disclosure: R. Bessho: None. Y. Takiyama: None. T. Takiyama: None. T. Ota: None. [ABSTRACT FROM AUTHOR]

The glomerular number and size are major factors in predicting outcomes in renal disease. We have recently established synchrotron radiation microcomputed tomography (SRµCT) imaging of the glomeruli in perfused kidneys, and reported the glomerular and renal hypertrophy, but not glomerular loss in obese type 2 diabetic db/db mice. To visualize pathophysiological basis of diabetic kidney of type 1 diabetes, we here explored SRµCT imaging of the glomeruli in streptozotocin-induced type 1 diabetic male mice (STZ mice) and of control male C57BL/6J mice (control mice). STZ mice at the age of 22 weeks significantly showed hyperglycemia and elevated urinary albumin excretion, but not hypertension or body weight changes compared to control mice. There was approximately 22% loss of the total glomerular number (Nglom) of STZ mice compared to that of control mice (p<0.001). STZ mice had fewer glomerular number in all renal cortical zone of the whole kidney than control mice (p=0.003). However, type 1 diabetes had no impact on the mean glomerular volume (Vglom), coefficient of variation (CV; SD/mean) of glomerular volume, and renal volume (Vkidney) (Vglom, p=0.149; CV, p=0.591; Vkidney, p=0.093 vs. control mice, respectively). Nglom was significantly correlated with body weight (r=0.458, p=0.032), blood glucose levels (r=-0.623, p=0.002) or HbA1c (r=-0.530, p=0.011). Vglom was correlated with Vkidney (r=0.622, p=0.002) by each other. Multivariate regression analysis with the stepwise forward method identified blood glucose (β=-0.665, p<0.001) and systolic blood pressure (β=-0.499, p=0.001) as significant independent determinants of Nglom. Our study, for the first time, demonstrated a significant reduction of the total glomerular number in STZ mice, without affecting the glomerular volume or renal volume. These results indicate the glomerular pathology markedly differs between type 1 and type 2 diabetes. Disclosure: Y. Takiyama: None. T. Sera: None. M. Nakamura: None. R. Bessho: None. T. Takiyama: None. T. Ota: None. M. Haneda: None. [ABSTRACT FROM AUTHOR]

The biguanide metformin has been used for treating insulin resistance and type 2 diabetes. Moreover, the UK prospective diabetes study (UKPDS) showed that metformin could reduce macrovascular morbility and mortality, suggesting anti-atherogenic and anti-inflammatory effects of metformin. However, it is not known whether metformin itself is able to reduce the risk for diabetic kidney disease like other insulin sensitizing agent, thiazolidinediones. The aim of present study is to determine the direct effect of metformin on the expression of the important molecules implicated in the progression of diabetic nephropathy in kidney cells in vitro. We used human renal proximal tubular epithelial cells (HRPTEC) because tubulo-interstitial injury was shown to play an important role in the progression of diabetic nephropathy. First, we identified the proteins secreted from HRPTEC exposed to 2.5 ng/ml TGF-β[sub 1], an important cytokine for the progression of diabetic nephropathy, using LC-MS/MS-based proteomic technique. Several proteins were identified to be upregulated by TGF-β[sub 1], one of which was plasminogen activator inhibitor-1 (PAI-1). In the Western blot analysis, TGF-β[sub 1]-induced PAI-1 protein secretion was significantly suppressed by the pretreatment of metformin in a dose-dependent fashion (0.1mM-10mM). We next examined the effect of metformin on the expression of genes induced by TGF-β[sub 1] by quantitative real-time RT-PCR. Metformin inhibited TGF-β[sub 1]-induced monocyte chemoattractant protein-1 (MCP-1), PAI-1, fibronectin, and collagen-1 mRNA expression. Moreover, metformin was also able to inhibit angiotensin II-induced MCP-1, collagen-1 mRNA expression. An activator of AMP-activated protein kinase (AMPK), which was known to be activated by metformin, 5-amino-4-imidazole carboxamide riboside (AICAR) also partially suppressed. MCP-1 mRNA expression. In conclusion, our results indicate that metformin not only inhibits TGF-β[sub 1]induced PAI-1 secretion, but also suppresses the expression of multiple genes linked to the progressive pathways in the diabetic nephropathy partly through the activation of AMPK. This action of metformin could be applied to prevent the progression of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is now a leading cause of end-stage renal disease. In addition, DN accounts for the increased mortality in type 1 and type 2 diabetes, and then patients without DN achieve long-term survival compatible with general population. Hypoxia represents an early event in the development and progression of DN, and hypoxia-inducible factor- (HIF-) 1 mediates the metabolic responses to renal hypoxia. Diabetes induces the "fraternal twins" of hypoxia, that is, pseudohypoxia and hypoxia. The kidneys are susceptible to hyperoxia because they accept 20% of the cardiac output. Therefore, the kidneys have specific vasculature to avoid hyperoxia, that is, AV oxygen shunting. The NAD-dependent histone deacetylases (HDACs) sirtuins are seven mammalian proteins, SIRTs 1-7, which are known to modulate longevity and metabolism. Recent studies demonstrated that some isoforms of sirtuins inhibit the activation of HIF by deacetylation or noncatalyzing effects. The kidneys, which have a vascular system that protects them against hyperoxia, unfortunately experience extraordinary hypernutrition today. Then, an unexpected overload of glucose augments the oxygen consumption, which ironically results in hypoxia. This review highlights the primary role of HIF in diabetic kidneys for the metabolic adaptation to diabetes-induced hypoxia. [ABSTRACT FROM AUTHOR]

Hyperalgesia is one of the symptoms of diabetic neuropathy, particularly in the early stages of diabetic neuropathy. The activation of nenrokinin1 (NK1) receptors in the spinal cord by substance P results in thermal and mechanical hyperalgesia. NK1 receptor expression is regulated by cAMP, activation of calcitonin gene-related peptide (CGRP) receptors, intracellular Ca2+, activation of N-methyl-D-aspartate (NMDA) receptors, and Ca2+/calmodulin-dependent protein kinase. We reported high glucose increases the expression of NK1 receptors in rat spinal cord cells. We investigated the mechanism of NK1 receptor expression induced by high glucose. We used primary cultures of neonatal rat spinal neurons to elucidate whether NK1 receptor expression is regulated by glucose. Using RT-PCR and Western blot analysis, it was determined that NK1 receptor expression increased in cells cultured under a high glucose condition. The high glucose-induced increase in NK1 receptor expression was not due to high osmotic pressure. Although inhibitors of protein kinase A, protein kinase C, and aldose reductase did not affect NK1 receptor expression, alpha-lipoic acid suppressed it under high glucose conditions. A specific inhibitor of mitochondrial complex II also suppressed the increase in NK1 receptor expression. Oxidative stress increased in cells cultured in high glucose, and this increase was suppressed by treatment with alpha-lipoic acid, as assessed by dihydroethidium fluorescence. These results indicate that high glucose increases NK1 receptor expression, which is due to oxidative stress. Antioxidants such as alpha-lipoic acid suppress the increase of NK1 receptors induced by high glucose. Since NK1 receptor activation contributes to increased excitability of spinal neurons, increased NK1 receptor expression may be important in maintaining the responsiveness of spinal neurons to substance P in central mechanisms underlying hyperalgesia. Administration of alpha-lipoic acid can become treatment for hyperalgesia in diabetic neuropathy. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy is characterized histologically by an accumulation of extracellular matrix proteins in the glomeruli. Such change in the glomeruli is thought to be associated with metabolic abnormalities in glomerolar mesangial cells (MC) specific to diabetes. Recent studies have shown that high glucose evokes a variety of signaling pathways in MC leading to dysregulation of gene expression and cellular function, however, precise mechanisms remain to be elucidated. The HIFs are transcription factors regulating gene expression in cellular adaptive response to hypoxic conditions. Since a fraction of target genes of the HIFs includes the genes encoding glycolytic enzymes, glucose transporters (GLUTs), and cell survival/death regulators, HIFs have been considered to play a role in regulation of energy metabolism and functions of the cells not only under hypoxic conditions but also in certain circumstances such as metabolic disturbances. In the present study, we examined the effect of ambient glucose levels on the expression of HIFs and subsequent target genes in cultured human MC. High glucose (HG, 25 mM) enhances expression of HIF-lα and HIF-2α proteins in MC under both normoxic and hypoxic conditions. Such effect of HG on HIFs expression was not observed in renal tubular epithelial cells and HeLa cells, suggesting a cell-type specificity of the effect of HG. In addition, HG induces expression of target genes of HIFs including GLUTs, and growth factors even under normoxic conditions, indicating a perturbation of HIFs-mediated regulation of the cellular functions by HG. On the other hand, addition of hydrogen peroxide or N-acetyl-L-cysteine to the cultures did not alter the basal and HG-induced level of HIFs, indicating that the effects of HG on the HIFs expression might not involve oxidant-dependent mechanism. In conclusion, HG alters HIF-mediated signal transduction in MC, which might contribute to dysregulatiuon of the glomerular function in diabetes. [ABSTRACT FROM AUTHOR]

High glucose evokes a variety of signals in mesangial cells that alter cellular functions responsible for the development of diabetic glomerulopathy. The hypoxia-inducible factor-1α (HIF-1α) regulates cellular homeostasis under hypoxic conditions, but it also has pleiotropic effects in response to cellular stresses at normoxia. Here we determined whether HIF-1α has a role in the regulation of mesangial cells in hyperglycemia. In the streptozotocin-induced diabetic mouse model, glomerular mesangial cells had a significant increase in HIF-1α expression in the nucleus. In cultured mesangial cells, high glucose enhanced the expression of HIF-1α and its target genes known to be involved in the development of diabetic glomerulopathy. A glucose-responsive carbohydrate response element binding protein (ChREBP) was found to have a critical role in the transcriptional upregulation of HIF-1α and downstream gene expression in mesangial cells exposed to high glucose. Knockdown of HIF-1α or ChREBP in mesangial cells abrogated the high glucose-mediated perturbation of gene expression. Our results show that ChREBP and HIF-1α mediate gene regulation in mesangial cells. Further studies will be needed to find out whether these findings are relevant to the development of the diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Abstract: Dendritic cells (DCs) are considered to be the most powerful antigen-presenting cells (APCs). DCs are thought to be associated with Th1 or Th2 polarization and with polarization-induced disease such as atopic dermatitis, asthma and allergic rhinitis, but its mechanism is not well known. In this study, we analyzed the mRNA expression of DCs between birch pollen allergic rhinitis and healthy controls by using cDNA array. We found that the expressions of CCL22/macrophage-derived chemokine (MDC) differed significantly. We also revealed that CCL22/MDC production was higher in patients than in healthy donors. By chemotaxis assay, CCL22/MDC can enhance the migration of patient’s T cells rather than those of healthy controls. Surface marker analysis of migrated cells revealed that the most of migrated cells expressed CCR4, which were considered to be Th2 cells. Furthermore, CD1a+ CD83+ cells located in the nasal mucosa expressed CCL22/MDC in vivo. To the best of our knowledge, this is the first report clearly indicating the role of CCL22/MDC in allergic rhinitis. [Copyright &y& Elsevier]

Visinin-like protein-3, which is one of the neuronal calcium sensors, has been shown to be mainly expressed in cerebellar Purkinje cells, but cellular function of this protein has not yet been elucidated. We examined the tissue distribution of murine visinin-like protein-3 transcripts using real-time reverse transcription-PCR. Visinin-like protein-3 mRNA was found to be expressed in peripheral tissues. Particularly, the expression of the transcript in the thymus was significantly higher than in other peripheral tissues. In addition, B6RVTC1 thymoma cells robustly expressed visinin-like protein-3 mRNA. To identify a target protein of visinin-like protein-3, we performed a pull-down experiment using glutathione S-transferase-tagged visinin-like protein-3 and two-dimensional electrophoresis. We demonstrated that microsomal cytochrome b5 was Ca2+-dependent binding partner of visinin-like protein-3. In a co-immunoprecipitation experiment, it was observed that hippocalcin, as well as visinin-like protein-3, could interact with cytochrome b5. Furthermore, we confirmed that the sequence Val114-Tyr127 at the C-terminal tail of cytochrome b5 is the minimal structural requirement for binding to visinin-like protein-3. In addition, the loop His19-His25 at the N terminus of visinin-like protein-3 is essential for binding to cytochrome b5. Microsomal cytochrome b5 was also shown to be a potential activator of cytochrome P450. The present findings raise the possibility that visinin-like protein-3 may link Ca2+ signal. ing to the machinery of microsomal monooxygenase complex composed of cytochrome b5, cytochrome P450, and some reductases. This report provides the first evidence of an interaction between visinin-like protein-3 and microsomal cytochrome b5. [ABSTRACT FROM AUTHOR]