Postprandial fatty acid-binding protein 4 is associated with muscle insulin resistance.

AIMS/HYPOTHESIS: Fatty acid-binding protein 4 (FABP4) has been reported to act as a hepatic insulin resistance factor. We previously reported that fasting FABP4 was correlated with insulin resistance measurements derived from the glucose clamp, and another study reported that postprandial FABP4 levels were decreased in healthy volunteers but were not reported (or known) in participants with type 2 diabetes. We have limited knowledge about the direct effect of FABP4 on muscle cells. We investigated the postprandial FABP4 levels in participants with type 2 diabetes, and the basic mechanism of muscle insulin resistance and FABP4. METHODS: We performed a meal tolerance test and hyperinsulinaemic-euglycaemic clamp in 22 participants with type 2 diabetes and 26 participants without diabetes. We measured fasting and postprandial serum FABP4. We cultured mouse C2C12 muscle cells, and investigated the effect of FABP4 on glucose uptake. We analysed insulin signalling by western blot and insulin binding assay. RESULTS: The postprandial FABP4 level in participants with type 2 diabetes was higher than that in participants without diabetes. Participants without diabetes had lower postprandial FABP4 than fasting except for one participant, whereas one-third of participants with type 2 diabetes had higher postprandial FABP4 than fasting. Postprandial FABP4 was correlated with the muscle insulin resistance M/I value from a glucose clamp in participants without diabetes (r=-0.42, p<0.05). The increase in FABP4 after a meal correlated with the muscle insulin resistance M/I value (r=-0.44, p<0.05) and the difference between fasting and postprandial glucagon in participants with type 2 diabetes (r=0.36, p<0.05). FABP4 alone appears to increase glucose uptake, and the combination of FABP4 and insulin decreases glucose uptake when compared with insulin alone. FABP4 inhibits insulin signalling of muscle cells through decreases in phosphorylation of insulin receptor substrate 1 and Akt. The physiological concentration of FABP4 did not inhibit insulin binding to muscle cells. CONCLUSIONS/INTERPRETATION: These results suggested that the postprandial FABP4 level is associated with insulin resistance, and FABP4 may suppress insulin signals.

Analysis of an antioxidative defence system of hydrogen peroxide-treated pancreatic islet-derived 1.1B4 cells and siRNA targeting NR4A3-treated cells by microarray.

Pancreatic islet ß-cells weaken under oxidative stress. In this study, human pancreatic islet-derived 1.1B4 cells were exposed to H2O2 and analysed using a human microarray, which revealed that heme oxygenase 1 (HMOX1), glutamate-cysteine ligase, early growth response 1, nuclear receptor subfamily 4 group A member 3 (NR4A3) and jun B proto-oncogene were upregulated, whereas superoxide dismutase 1 and catalase were not. Expression of NR4A3 rapidly increased after H2O2 addition, and the 1.1B4 cells treated with siRNA targeting NR4A3 became sensitive to H2O2; further, HMOX1 expression was strongly inhibited, suggesting that NR4A3 is an oxidative stress-responsive transcription factor that functions through HMOX1 expression in pancreatic islet ß-cells. Expression of cyclin E1 and cyclin-dependent kinase 1 was also inhibited by siRNAs targeting NR4A3.

Aldehyde dehydrogenase 2 polymorphism is an important gene for insulin resistance in Japanese patients with type 2 diabetes.

Background: Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme involved in alcohol metabolism. ALDH2 polymorphism has been reported as a risk factor for type 2 diabetes mellitus (T2DM) and is associated with liver insulin resistance due to alcohol consumption in non-diabetic individuals. Herein, we investigated the association between ALDH2 polymorphisms and insulin resistance in patients with T2DM. Methods: We performed a meal tolerance test and the hyperinsulinemic-euglycemic clamp on 71 Japanese participants: 34 patients with T2DM, and 37 non-diabetic participants. We analyzed the ALDH2 polymorphism (ALDH2 rs67); GG type was defined as the T2DM high-risk group, compared with the low-risk AG and AA groups. Results: Glucose levels were similar in the high- and low-risk T2DM groups. The high-risk group for T2DM showed a significantly higher BMI (p < 0.005), insulin resistance in HOMA-IR (p < 0.05), and Insulin sensitivity index (p < 0.05); however, there were no significant differences in insulin resistance in the clamp test (p = 0.10). Alcohol consumption did not differ significantly between groups (p = 0.66). Non-diabetic participants also showed higher HOMA-IR insulin resistance in the high-risk group (p < 0.05), but insulin resistance levels in the glucose clamp tests (p = 0.56) and insulin secretion were not significant. Conclusion: The results suggest that ALDH2 is an important gene associated with insulin resistance and obesity in Japanese patients with type 2 diabetes.

Fasting hepatic insulin clearance reflects postprandial hepatic insulin clearance: a brief report.

BACKGROUND: Hepatic insulin clearance (HIC) is an important pathophysiology of type 2 diabetes mellitus (T2DM). HIC was reported to decrease in patients with type 2 diabetes and metabolic syndrome. HIC is originally calculated by post-load insulin and C-peptide from the oral glucose tolerance test (OGTT). However, OGTT or meal tolerance tests are a burden for patients, and OGTT is not suitable for overt diabetes due to the risk of hyperglycemia. If we can calculate the HIC from the fasting state, it is preferable. We hypothesized that fasting HIC correlates with postprandial HIC in both participants with T2DM and without diabetes. We investigated whether fasting HIC correlates with postprandial HIC in overt T2DM and nondiabetes subjects (non-DM) evaluated by using glucose clamp and meal load. METHODS: We performed a meal tolerance test and hyperinsulinemic-euglycemic clamp in 70 subjects, 31 patients with T2DM and 39 non-DM subjects. We calculated the postprandial C-peptide AUC-to-insulin AUC ratio as the postprandial HIC and the fasting C-peptide-to-insulin ratio as the fasting HIC. We also calculated whole-body insulin clearance from the glucose clamp test. RESULTS: The fasting HIC significantly correlated with postprandial HIC in T2DM (r_S = 0.82, P < 0.001). Nondiabetes subjects also showed a significant correlation between fasting and postprandial HIC (r_S = 0.71, P < 0.001). Fasting HIC in T2DM was correlated with BMI, HbA1c, gamma-glutamyl transpeptidase, HOMA-IR, HOMA-beta, M/I, and whole-body insulin clearance. Fasting HIC in nondiabetes subjects was correlated with HOMA-IR and HOMA-beta. CONCLUSIONS: These results suggest that fasting HIC is strongly correlated with postprandial HIC in both overt T2DM and non-DM patients, as evaluated by the meal test and glucose clamp method. Fasting HIC could be a convenient marker of HIC.

The sodium-glucose cotransporter 2 inhibitor ipragliflozin improves liver function and insulin resistance in Japanese patients with type 2 diabetes.

Sodium-glucose cotransporter 2 inhibitor (SGLT2i) treatment is a therapeutic approach for type 2 diabetes mellitus (T2DM). Some reports have shown that SGLT2i treatment improves insulin resistance; however, few studies have evaluated insulin resistance by the glucose clamp method. Hepatic insulin clearance (HIC) is a new pathophysiological mechanism of T2DM. The effect of SGLT2i treatment on hepatic insulin clearance and insulin resistance is not well known. We investigated the effect of SGLT2i treatment on insulin resistance, insulin secretion, incretin levels, body composition, and hepatic insulin clearance. We conducted a meal tolerance test (MTT) and a hyperinsulinemic-euglycemic clamp test in 9 T2DM patients. Ipragliflozin (50 mg/day) was administered, and the MTT and clamp test were performed after 4 months. We calculated HIC as the postprandial C-peptide AUC-to-insulin AUC ratio. We also measured GLP-1, GIP, and glucagon levels during the MTT. Body weight and HbA1c were decreased, although not significantly, after 4 months of treatment. Postprandial glucose, fasting insulin and postprandial insulin were significantly decreased. Insulin resistance with the glucose clamp was not changed, but the HOMA-IR and insulin sensitivity indices were significantly improved. Incretin and glucagon levels were not changed. Hepatic insulin clearance was significantly increased, but whole-body insulin clearance was not changed. The FIB-4 index and fatty liver index were significantly reduced. The HOMA-beta and insulinogenic indices were not changed, but the C-peptide index was significantly increased. Although the number of patients was small, these results suggested that SGLT2i treatment improved liver function, decreased hepatic insulin resistance, and increased hepatic insulin clearance, despite the small weight reduction.

Significance of pancreatic duodenal homeobox-1 (PDX-1) genetic polymorphism in insulin secretion in Japanese patients with type 2 diabetes.

INTRODUCTION: Pancreatic and duodenal homeobox factor-1 (PDX-1) is an imperative gene for insulin secretion in maturity-onset diabetes of the young 4. PDX-1 gene polymorphism was associated with lower first-phase insulin secretion in a genome-wide association study of intravenous glucose tolerance test. It was not associated with type 2 diabetes risk and insulin secretion in a genome-wide oral glucose tolerance test study. However, there have been no reports of overt type 2 diabetes and insulin resistance evaluation using a glucose clamp. We investigated PDX-1 polymorphism, insulin secretion, and insulin resistance in overt type 2 diabetes. RESEARCH DESIGN AND METHODS: We performed a meal tolerance test (MTT) and hyperinsulinemic-euglycemic clamping on 63 Japanese subjects, 30 with type 2 diabetes and 33 non-diabetic. We analyzed the rs1124607 PDX-1 gene polymorphism and defined A/C and C/C as the high-risk group and A/A as the low-risk group. RESULTS: HOMA-beta (homeostatic model assessment beta-cell function) was significantly lower in the high-risk group than in the low-risk group for all subjects (72.9±54.2% vs 107.0±63.5%, p<0.05). Glucose levels and glucose area under the curve (AUC) were not significantly different between both the risk groups. The insulin levels at 60 and 120 min and the insulin AUC after MTT were remarkably lower in the high-risk group than those in the low-risk group for all subjects (AUC 75.7±36.7 vs 112.7±59.5, p<0.05). High-risk subjects with type 2 diabetes had significantly lower insulin levels at 30 and 60 min and insulin AUC than low-risk subjects. Non-diabetic high-risk subjects depicted significantly lower insulin levels at 120 and 180 min. There were negligible differences in insulin resistance between the risk groups. CONCLUSIONS: These results suggest that the PDX-1 genetic polymorphism is crucial for insulin secretion in Japanese patients with type 2 diabetes.

Dipeptidyl peptidase 4 inhibitor improves insulin resistance in Japanese patients with type 2 diabetes: a single-arm study, a brief report.

BACKGROUND: Dipeptidyl peptidase 4 inhibitor (DPP4i) is an effective medicine for type 2 diabetes mellitus (T2DM). Some articles reported DPP4i improves insulin secretion and insulin resistance. However, these effects are not well established by glucose clamp test and test meal in Japanese. We investigated the effect of DPP4i on insulin resistance and insulin secretion by using the glucose clamp test and meal tolerance test (MTT). METHODS: We performed a MTT, and the hyperinsulinemic-euglycemic clamp in 8 Japanese patients with T2DM. This study was a single-arm study. We measured fasting and postprandial glucose, insulin, incretins, and glucagon levels. We also measured serum adiponectin levels. RESULTS: HbA1c was significantly decreased after 3 months. The fasting and postprandial glucose levels were significantly decreased. Fasting and postprandial insulin levels were not changed. The insulin resistance derived from the glucose clamp test was significantly improved. HOMA-IR was not significantly changed. GLP-1 and GIP were significantly increased but glucagon did not change. Adiponectin was not significantly changed. CONCLUSIONS: Although the number of patients was very small, these results suggested that DPP4i treatment might improve insulin resistance without changing insulin secretion.

Dose of 3-methylcholanthrene enhances vitamin C accumulation and mRNA expression of its transporter in the liver of ODS rats and in HepG2 cells.

Polycyclic aromatic hydrocarbon (PAH) compounds including 3-methylcholanthrene induce harmful reactive intermediates and reactive oxygen species. This study reports the effect of 3-methylcholanthrene on the accumulation of vitamin C and the expression of vitamin C transporters. ODS rats were given l-ascorbic acid daily and intraperitoneal injections of 10 mg 3-methylcholanthrene in total. On day 10, vitamin C concentrations and the expression of vitamin C transporter in the tissues were measured. As a result, the levels of sodium-dependent vitamin C transporter (SVCTs) 1 and the l-ascorbic acid concentration in 3-methylcholanthrene-treated livers and hepatocytes have increased significantly. However, the content of vitamin C in the urine and TBARS in the liver have not changed. These results suggest that the administration of 3-methylcholanthrene elevates the requirement for vitamin C via (SVCTs) 1 due to xenobitics-metabolizing, such as the induction of cytochrome P450 family.

The level of orally ingested vitamin C affected the expression of vitamin C transporters and vitamin C accumulation in the livers of ODS rats.

We investigated the effects of vitamin C administration on vitamin C-specific transporters in ODS/ShiJcl-od/od rat livers. The vitamin C-specific transporter levels increased in the livers of the rats not administered vitamin C and decreased in the livers of those administered vitamin C at 100 mg/d, indicating that these transporter levels can be influenced by the amount of vitamin C administered.

Free advanced glycation end product distribution in blood components and the effect of genetic polymorphisms.

One hypothesis regarding the cause of diabetic complications is that advanced glycation end products (AGEs) bind to the AGE receptor and induce changes in gene expression. However, what AGEs exist in vivo and how individual AGEs are produced and impact body metabolic process to cause diabetes complications are not understood. We developed a new precise method to measure AGEs using LC-MS/MS with a new column and measured 7 free AGEs, including N(6)-carboxymethyllysine (CML), N(6)-(1-carboxyethyl)-l-lysine (CEL) and N5-(5-hydro-5-methyl-4-imidazolon-2-yl)L-ornithine (MG-H1), in human blood components. Blood was obtained from 9 people, and free AGEs were measured in individual blood components with LC-MS/MS before and after a meal. Free CML and CEL were abundant in erythrocytes, with 92% of free CML and 85% of free CEL localized in erythrocytes. In contrast, 60% of free MG-H1 was distributed in the serum. After the meal, free serum MG-H1 increased, but CML and CEL did not. CML and CEL are mainly distributed in erythrocytes and were not affected by the meal, indicating that they are produced in vivo. However, the main source of MG-H1 is the meal. The effect of genetic polymorphisms on AGEs was also investigated. Low activity type aldehyde dehydrogenase 2 (ALDH2) increased the CML concentration in the blood. This is the first observation that shows that the metabolic process of CML and CEL is different from that of MG-H1 and the effect of ALDH2 SNPs on CML.

Hepatic insulin clearance is increased in patients with high HbA1c type 2 diabetes: a preliminary report.

INTRODUCTION: Hepatic insulin clearance (HIC) is an important pathophysiology of type 2 diabetes. HIC was reported to decrease in patients with type 2 diabetes and metabolic syndrome. However, hyperglycemia was suggested to enhance HIC, and it is not known whether poorly controlled diabetes increases HIC in patients with type 2 diabetes. We investigated whether HIC was increased in patients with poorly controlled diabetes, and whether HIC was associated with insulin resistance and incretins. RESEARCH DESIGN AND METHODS: We performed a meal tolerance test and the hyperinsulinemic-euglycemic clamp in 21 patients with type 2 diabetes. We calculated the postprandial C-peptide area under the curve (AUC)-to-insulin AUC ratio as the HIC; measured fasting and postprandial glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon levels and analyzed serum adiponectin and zinc transporter-8 (ZnT8) gene polymorphism. RESULTS: The HIC significantly correlated with glycated hemoglobin (HbA1c) (r_S=0.58, p<0.01). In patients with high HIC above the median of 6.5, the mean HbA1c was significantly higher compared with low HIC below the median. Homeostatic model assessment (HOMA)-beta (r_S=-0.77, p<0.01) and HOMA-IR (r_S=-0.66, p<0.005) were correlated with HIC. The M/I value in the clamp study was correlated with HIC. GLP-1-AUC and GIP-AUC were not correlated with HIC. Glucagon-AUC was negatively correlated with HIC, but there were no significant differences between the high and low HIC groups. Adiponectin was positively correlated with HIC. The ZnT8 gene polymorphism did not affect HIC. CONCLUSIONS: These results suggest that HIC was increased in patients with high HbA1c type 2 diabetes, low insulin secretion, low insulin resistance and high adiponectin conditions.

Correction: Serum fatty acid-binding protein 4 (FABP4) concentration is associated with insulin resistance in peripheral tissues, A clinical study.

[This corrects the article DOI: 10.1371/journal.pone.0179737.].

Altered signaling pathway in the dysmorphogenesis of telencephalon in the Gli3 depressed mouse embryo, Pdn/Pdn.

The responsible gene of genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) is Gli3. Pdn/Pdn exhibits absence of the olfactory bulb, suggesting telencephalic dysmorphogenesis. It has been cleared that a transposon was inserted into intron 3 of the Gli3 gene in the Pdn mouse. Adequate PCR primers in the intron 3 and transposon allowed us to discriminate +/+, Pdn/+ and Pdn/Pdn embryos. After genotyping of the Pdn embryos using genomic DNA from the yolk sac membrane, gene expressions in the embryo proper were analyzed by DNA microarray, real-time PCR and whole-mount in situ hybridization (WISH) methods. DNA microarray detected 368 depressed and 425 over-expressed genes in the Pdn/Pdn mouse embryos on day 9 of gestation. In these genes, six signaling pathway and 20 transcription factor genes were included. From these genes, we further investigated Gli3, Emx2, Wnt8b and Wnt7b gene expressions using real-time PCR and WISH, and depression of these gene expression amounts and altered expression patterns were confirmed. Although alterations of Shh and Fgf8 gene expressions were not detected in the DNA microarray, as these genes have been closed up in the telencephalic morphogenesis, we investigated these gene expressions by real-time PCR and WISH. Shh gene expression amount and pattern were not changed. Alteration of Fgf8 gene expression amount was not detected also in the real-time PCR, but altered expression pattern was detected in the Pdn/Pdn embryos by WISH. From the present data, we suggested that Emx2, Wnt8b, Wnt7b and Fgf8 are the important Gli3 signaling pathway in the morphogenesis of telencephalon.

CPR-IR is an insulin resistance index that is minimally affected by hepatic insulin clearance-A preliminary research.

BACKGROUND: Increased hepatic insulin clearance (HIC) is important in the pathophysiology of type 2 diabetes mellitus (T2DM). The aim of this study is to analyze an effective insulin resistance (IR) index that is minimally affected by HIC. METHODS: Our study involved 20 participants with T2DM and 21 healthy participants without diabetes (Non-DM). Participants underwent a meal tolerance test from which plasma glucose, insulin and serum C-peptide immunoreactivity (CPR) were measured, and HOMA-IR and HIC were calculated. Participants then underwent a hyperinsulinemic-euglycemic clamp from which the glucose disposal rate (GDR) was measured. RESULTS: The index CPR-IR = 20/(fasting CPR × fasting plasma glucose) was correlated more strongly with GDR, than was HOMA-IR, and CPR-IR could be used to estimate GDR. In T2DM participants with HIC below the median, HOMA-IR and CPR-IR were equally well correlated with GDR. In T2DM with high HIC, CPR-IR correlated with GDR while HOMA-IR did not. In Non-DM, CPR-IR and HOMA-IR were equally well correlated with GDR regardless of HIC. The mean HIC value in T2DM was significantly higher than that of Non-DM. CONCLUSIONS: CPR-IR could be a simple and effective index of insulin resistance for patients with type 2 diabetes that is minimally affected by HIC.

Body mass index ≥23 is a risk factor for insulin resistance and diabetes in Japanese people: A brief report.

BACKGROUND: Screening for undiagnosed type 2 diabetes mellitus is recommended for Asian Americans with a body mass index ≥23. However, the optimal body mass index cut-off score for predicting the risk of diabetes mellitus in Japanese people is not well known. The aim of this study was to determine the best body mass index cut-off score for predicting insulin resistance and diabetes mellitus in the Japanese population. METHODS: This study had two parts, a clinical investigation and a retrospective observational investigation. In the clinical part of the study, 58 participants (26 with type 2 diabetes mellitus and 32 non-diabetics) underwent a hyperinsulinemic-euglycemic clamp from which their glucose disposal rate was measured. For the retrospective part of the study, medical check-up data from 88,305 people in the Tottori Prefecture were analyzed for clinical evidence of diabetes mellitus. The optimal BMI cut-off scores for prediction of insulin resistance and diabetes mellitus were determined. RESULTS: In the clamp study, the optimal body mass index cut-off score to predict insulin resistance in non-diabetic patients was 22.7. All participants with type 2 diabetes mellitus were insulin resistant, and the optimal body mass index cut-off score for prediction of severe insulin resistance was 26.2. When the data from the type 2 diabetic and the non-diabetic participants were combined, the optimal body mass index cut-off score for prediction of insulin resistance was 23.5. Analysis of 88,305 medical check-up records yielded an optimal body mass index cut-off score for prediction of diabetes mellitus of 23.6. CONCLUSIONS: These results suggest that having a body mass index ≥23 is a risk factor for insulin resistance and diabetes mellitus in the Japanese population.

Prevention of ochratoxin A-induced neural tube defects by folic acid in the genetic polydactyly/arhinencephaly mouse, Pdn/Pdn.

The gene responsible for the polydactyly/arhinencephaly (Pdn/Pdn) mouse, which exhibits polysyndactyly and arhinencephaly and has a 13.2% risk of neural tube defects (NTD), has been identified as Gli3. Ochratoxin A (OTA) is a teratogen causing NTD in mice. When Pdn/Pdn embryos were exposed to 2 mg/kg of OTA on day 7.5, the incidence of NTD in Pdn/Pdn fetuses increased to 51.6%. Pre-treatment with folinic acid (FA), metabolically the most active form of folic acid, before OTA-treatment decreased the incidence of NTD to 20.8%. We investigated the effect of OTA and FA on gene expression in day 9 embryos using whole-mount in situ hybridization and real-time PCR. Over-expression of Fgf8 was observed at the anterior neural ridge (ANR) in the non-treated Pdn/Pdn. Over-expression at the ANR expanded in the OTA-treated Pdn/Pdn, and it was ameliorated by pretreatment with FA. Emx2 signal was observed in the dorsal forebrain in the non-treated +/+, but disappeared in the OTA-treated +/+, and was recovered by FA. The Emx2 signal was pale and the expression amount was depressed in the non-treated and OTA-treated Pdn/Pdn embryos. It was suggested that down-regulation of Gli3 induced the over-expression of Fgf8 at the ANR, that OTA treatment accelerated the over-expression, and that pretreatment with FA ameliorated the OTA-induced over-expression of Fgf8 in the Pdn/Pdn. It was also suggested that down-regulation of Gli3 induced the down-regulation of Emx2 in the Pdn/Pdn. It was further speculated that the over-expression of Fgf8 at the ANR and down-regulation of Emx2 in the dorsal forebrain may contribute to NTD induction.

Serum fatty acid-binding protein 4 (FABP4) concentration is associated with insulin resistance in peripheral tissues, A clinical study.

Type 2 diabetes mellitus (T2DM) is caused by insulin resistance and ß cell dysfunction. In recent studies reported that several markers associated with insulin sensitivity in skeletal muscle, Adiponectin and other parameters, such as fatty acid-binding protein (FABP4), have been reported to regulate insulin resistance, but it remains unclear which factor mostly affects insulin resistance in T2DM. In this cross-sectional study, we evaluated the relationships between several kinds of biomarkers and insulin resistance, and insulin secretion in T2DM and healthy controls. We recruited 30 participants (12 T2DM and 18 non-diabetic healthy controls). Participants underwent a meal tolerance test during which plasma glucose, insulin and serum C-peptide immunoreactivity were measured. We performed a hyperinsulinemic-euglycemic clamp and measured the glucose-disposal rate (GDR). The fasting serum levels of adiponectin, insulin-like growth factor-1, irisin, autotaxin, FABP4 and interleukin-6 were measured by ELISA. We found a strong negative correlation between FABP4 concentration and GDR in T2DM (r = -0.657, p = 0.020). FABP4 also was positively correlated with insulin secretion during the meal tolerance test in T2DM (IRI (120): r = 0.604, p = 0.038) and was positively related to the insulinogenic index in non-DM subjects (r = 0.536, p = 0.022). Autotaxin was also related to GDR. However, there was no relationship with insulin secretion. We found that serum FABP4 concentration were associated with insulin resistance and secretion in T2DM. This suggests that FABP4 may play an important role in glucose homeostasis.

High Serum Advanced Glycation End Products Are Associated with Decreased Insulin Secretion in Patients with Type 2 Diabetes: A Brief Report.

OBJECTIVE: Advanced glycation end products (AGEs) are important in the pathophysiology of type 2 diabetes mellitus (T2DM). They directly cause insulin secretory defects in animal and cell culture models and may promote insulin resistance in nondiabetic subjects. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry method for measuring AGEs in human serum. Here, we use this method to investigate the relationship between AGEs and insulin secretion and resistance in patients with T2DM. METHODS: Our study involved 15 participants with T2DM not on medication and 20 nondiabetic healthy participants. We measured the AGE carboxyethyllysine (CEL), carboxymethyllysine (CML), and methyl-glyoxal-hydro-imidazolone (MG-H1). Plasma glucose and insulin were measured in these participants during a meal tolerance test, and the glucose disposal rate was measured during a euglycemic-hyperinsulinemic clamp. RESULTS: CML and CEL levels were significantly higher in T2DM than non-DM participants. CML showed a significant negative correlation with insulin secretion, HOMA-%B, and a significant positive correlation with the insulin sensitivity index in T2DM participants. There was no correlation between any of the AGEs measured and glucose disposal rate. CONCLUSIONS: These results suggest that AGE might play a role in the development or prediction of insulin secretory defects in type 2 diabetes.

Genetic susceptibility in the neural tube defects induced by ochratoxin A in the genetic arhinencephaly mouse, Pdn/Pdn.

It is well known that ochratoxin A (OTA) induces neural tube defects (NTDs) in mice. In the present study, OTA was administered to the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) to investigate the synergistic effect between gene and environmental toxin. OTA treatment on day 7.5 of gestation increased NTDs in the Pdn/Pdn mouse. The responsible gene for Pdn/Pdn is Gli3. So, it was speculated that specific susceptibility for OTA in the Pdn/Pdn mouse embryo may be due to the severe depression of Gli3 gene expression. As correlated genes, Gli3, Shh and Fgf8 gene expressions were examined in the Pdn mouse embryo on day 9 of gestation after administration of OTA on day 7.5. No alteration of Shh expression was observed in the non-treated Pdn/Pdn, and OTA-treated +/+ and Pdn/Pdn. Fgf8 signal was observed at the anterior neural ridge (ANR) in the non-treated +/+, and that was elongated in the non-treated Pdn/Pdn, and further elongated and more intensive in the OTA-treated Pdn/Pdn. It was suggested that Fgf8 gene expression was affected by the depression of Gli3, and alteration of Fgf8 gene expression was accelerated by the toxicity of OTA in the Pdn/Pdn.

Simultaneous Quantitation of Advanced Glycation End Products in Soy Sauce and Beer by Liquid Chromatography-Tandem Mass Spectrometry without Ion-Pair Reagents and Derivatization.

The aim of this study was to develop a simple and sensitive method to analyze several advanced glycation end products (AGEs) simultaneously using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to apply this method to the quantitation of AGEs in brown-colored foods. The developed method enabled to separate and quantitate simultaneously seven AGEs, and was applied to the determination of free AGEs contained in various kinds of soy sauce and beer. The major AGEs in soy sauce and beer were Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (MG-H1). Using the developed LC-MS/MS method, recovery test on soy sauce and beer samples showed the recovery values of 85.3-103.9% for CML, 95.9-107.4% for CEL, and 69.5-123.2% for MG-H1. In particular, it is the first report that free CML, CEL, and MG-H1 were present in beer. Furthermore, long-term storage and heating process of soy sauce increased CML and MG-H1.