Iron loading impairs lipoprotein lipase activity and promotes hypertriglyceridemia.

Iron loading is associated with altered lipid metabolism, but underlying mechanisms remain unknown. We compared serum iron and triglycerides (TGs) in Belgrade rats, a genetic model of iron-loading anemia. Homozygous b/b rats had greater serum iron (68 vs. 28 µM; P=0.0004) and TG levels (180 vs. 84 mg/dl; P=0.014) compared to +/b controls. To confirm the association between iron loading and high TGs, Fischer rats were fed chow containing 1% carbonyl iron. Compared to controls pair-fed normal chow, carbonyl iron-fed rats had elevated serum iron (42 vs. 21 µM; P=0.007) and TGs (190 vs. 115 mg/dl; P=0.009). Despite normal hepatic production and secretion, TG clearance was lower in b/b than +/b rats due to reduced serum lipoprotein lipase (LPL) activity (3.1 vs. 5.0 mM/min; P=0.026). Likewise, LPL was lower in carbonyl iron-fed rats compared to controls (2.4 vs. 3.7 mM/min; P=0.017). Direct addition of iron to serum ex vivo or recombinant LPL in vitro decreased enzymatic activity in a dose-dependent manner. Lowering serum iron in Belgrade rats reduced TG levels (274 to 67 mg/dl, P=0.001). This study explains the relationship between iron status and lipid metabolism and provides mechanistic support for interventions that reduce serum iron levels in individuals at risk for hypertriglyceridemia.

Glucose metabolism in the Belgrade rat, a model of iron-loading anemia.

The iron-diabetes hypothesis proposes an association between iron overload and glucose metabolism that is supported by a number of epidemiological studies. The prevalence of type 2 diabetes in patients with hereditary hemochromatosis and iron-loading thalassemia supports this hypothesis. The Belgrade rat carries a mutation in the iron transporter divalent metal transporter 1 (DMT1) resulting in iron-loading anemia. In this study, we characterized the glycometabolic status of the Belgrade rat. Belgrade rats displayed normal glycemic control. Insulin signaling and secretion were not impaired, and pancreatic tissue did not incur damage despite high levels of nonheme iron. These findings suggest that loss of DMT1 protects against oxidative damage to the pancreas and helps to maintain insulin sensitivity despite iron overload. Belgrade rats had lower body weight but increased food consumption compared with heterozygous littermates. The unexpected energy balance was associated with increased urinary glucose output. Increased urinary excretion of electrolytes, including iron, was also observed. Histopathological evidence suggests that altered renal function is secondary to changes in kidney morphology, including glomerulosclerosis. Thus, loss of DMT1 appears to protect the pancreas from injury but damages the integrity of kidney structure and function.

Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.

Hydrogen sulfide (H(2)S) has been traditionally known for its toxic effects on living organisms. The role of H(2)S in the homeostatic regulation of pancreatic insulin metabolism has been unclear. The present study is aimed at elucidating the effect of endogenously produced H(2)S on pancreatic insulin release and its role in diabetes development. Diabetes development in Zucker diabetic fatty (ZDF) rats was evaluated in comparison with Zucker fatty (ZF) and Zucker lean (ZL) rats. Pancreatic H(2)S production and insulin release were also assayed. It was found that H(2)S was generated in rat pancreas islets, catalyzed predominantly by cystathionine gamma-lyase (CSE). Pancreatic CSE expression and H(2)S production were greater in ZDF rats than in ZF or ZL rats. ZDF rats exhibited reduced serum insulin level, hyperglycemia, and insulin resistance. Inhibition of pancreatic H(2)S production in ZDF rats by intraperitoneal injection of DL-propargylglycine (PPG) for 4 weeks increased serum insulin level, lowered hyperglycemia, and reduced hemoglobin A1c level (P<0.05). Although in ZF rats it also reduced pancreatic H(2)S production and serum H(2)S level, PPG treatment did not alter serum insulin and glucose level. Finally, H(2)S significantly increased K(ATP) channel activity in freshly isolated rat pancreatic beta-cells. It appears that insulin release is impaired in ZDF because of abnormally high pancreatic production of H(2)S. New therapeutic approach for diabetes management can be devised based on our observation by inhibiting endogenous H(2)S production from pancreas.

Attenuation of hypertension development by scavenging methylglyoxal in fructose-treated rats.

OBJECTIVES: Methylglyoxal is a reactive dicarbonyl intermediate of metabolism produced in the body. It reacts with certain proteins and forms damaging advanced glycation endproducts (AGEs) such as N epsilon-carboxyethyl-lysine (CEL) and N epsilon-carboxymethyl-lysine (CML). Increased methylglyoxal levels are found in diabetes mellitus and associated with hypertension development in the spontaneously hypertensive rats (SHR). The purpose of this study was to investigate whether increased endogenous formation of methylglyoxal and methylglyoxal-induced AGEs caused hypertension development in normotensive Sprague Dawley rats. METHODS: The rats were fed chronically for 16 weeks with fructose, a known precursor of methylglyoxal formation. One group of rats was cotreated with fructose and metformin, an AGEs formation inhibitor. Methylglyoxal and reduced glutathione (GSH) were measured by high performance liquid chromatography, whereas hydrogen peroxide was measured by a dicholorofluorescin assay. Immunohistochemistry was performed for endothelial nitric oxide synthase (eNOS), CEL and CML. RESULTS: Fructose-fed rats had elevated blood pressure, serum methylglyoxal and triglycerides and reduced serum levels of GSH. Methylglyoxal, hydrogen peroxide and CEL were increased in the aorta, whereas eNOS was reduced. CEL and CML were also increased in the mesenteric artery endothelium along with media/lumen ratio, signifying structural remodelling. All the harmful changes in fructose-fed rats were attenuated in metformin and fructose cotreated rats. CONCLUSION: Increased methylglyoxal, AGEs, oxidative stress and reduced eNOS along with structural remodeling of the vessel wall in the aorta and mesenteric artery likely play a role in the pathogenesis of hypertension.

Structural and functional changes in human insulin induced by methylglyoxal.

Elevated methylglyoxal (MG) levels have been reported in insulin-resistance syndrome. The present study investigated whether MG, a highly reactive metabolite of glucose, induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed on mass spectrometric (MS) analysis of the incubates. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after the treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentrations. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. MG alone had no effect on glucose uptake or the transcriptional expression of insulin receptor. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic beta-cells. The degradation of MG-insulin through liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in beta-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of insulin molecule may contribute to the pathogenesis of insulin resistance.

Efficacy and Safety of OnabotulinumtoxinA in Patients with Neurogenic Detrusor Overactivity: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Neurogenic detrusor overactivity (NDO) affects the quality of life (QoL) of millions of individuals worldwide. The purpose of this study was to assess the efficacy and safety of onabotulinumtoxinA in patients with NDO using a network meta-analytic approach, which can also quantify and compare the efficacy of onabotulinumtoxinA across different dosages. METHODS: PubMed, EMBASE, and the Controlled Trials Register were searched to identify randomized controlled trials comparing onabotulinumtoxinA to a control for NDO in adult patients. The primary outcome was the mean number of urinary incontinence (UI) episodes per week. Urodynamic parameters included the maximum cystometric capacity (MCC) and the maximum detrusor pressure (MDP). The safety of onabotulinumtoxinA was determined by the incidence of various frequent adverse events (AEs). Two authors extracted data independently, and the statistical analyses were performed using RevMan 5.1.0 software. RESULTS: A total of 1,915 patients from six randomized controlled trials were included in this meta-analysis. The onabotulinumtoxinA-treated groups had a significantly decreased mean number of urinary incontinence episodes per week (at week 6) (onabotulinumtoxinA200U: MD: -10.72, 95% CI: -13.4 to -8.04, P<0.00001; 300 U: MD: -11.42, 95% CI: -13.91 to -8.93, P<0.00001), MDP (200 U: MD: -33.46, 95% CI: -39.74 to -27.18, P<0.00001; 300 U: MD: -31.72, 95% CI: -37.69 to -25.75, P<0.00001), and greater increased MCC (200 U: MD: 141.30, 95% CI: 121.28 to 161.32, P<0.00001; 300 U: MD: 151.39, 95% CI: 130.43 to 172.34, P<0.00001) compared to the placebo-treated groups. However, there were no significant differences between the onabotulinumtoxinA-treated groups for the number of weekly UI episodes at 6 weeks (MD: 0.08, 95% CI: -2.57 to 2.73, P = 0.95). Similarly, we also observed that there were no significant differences in MCC (MD: -9.97, 95% CI: -33.15 to 13.20, P = 0.40) and MDP (MD: -1.86, 95% CI: -8.09 to 4.37, P = 0.56). Considering the AEs, the onabotulinumtoxinA-treated groups were often associated with more complications, including urinary tract infections (UTIs) (RR: 1.47, 95% CI: 1.29 to 1.67, P<0.00001), urinary retention (RR: 5.58, 95% CI: 3.53 to 8.83, P<0.00001), hematuria (RR: 1.70, 95% CI: 1.01 to 2.85, P = 0.05), and muscle weakness (RR: 2.59, 95% CI: 1.36 to 4.91, P = 0.004). CONCLUSIONS: OnabotulinumtoxinA can significantly reduce the frequency of urge urinary incontinence and improve urodynamic parameters (MCC and MDP) in patients with NDO at 6 weeks after treatment. This meta-analysis indicates that onabotulinumtoxinA is effective and safe for treating patients with NDO compared to placebo. Additionally, we did not observe any statistical or clinical differences in efficacy between 300 and 200 U dosages.

Compromised DNA repair enhances sensitivity of the yeast RNR3-lacZ genotoxicity testing system.

The RNR3-lacZ genotoxicity testing system was developed based on the induction of a Saccharomyces cerevisiae RNR3-lacZ reporter gene in response to a broad range of DNA-damaging agents. In order to enhance the sensitivity of the RNR3-lacZ system, several deletion mutant strains representing different repair pathways were created and examined for their effects on RNR3-lacZ expression. It was found that inactivation of different DNA repair pathways has profound effects on the DNA damage induction of RNR3 expression. Although deletion of MAG1 in the base excision repair pathway enhances the detection sensitivity to DNA-alkylating agents, and deletion of RAD2 in the nucleotide excision repair pathway enhances the detection sensitivity to ultraviolet and agents that produce bulky lesions, inactivation of genes involved in the recombination repair and postreplication repair variably reduces RNR3-lacZ induction. This study not only helps to establish a more sensitive genotoxicity testing system but also suggests that certain eukaryotic DNA repair pathways are required for gene regulation in response to DNA damage and probably serve as sensors in the signal transduction cascade.

A stable and sensitive genotoxic testing system based on DNA damage induced gene expression in Saccharomyces cerevisiae.

A sensitive and stable genotoxic testing system has been developed based on the induction of a Saccharomyces cerevisiae RNR3-lacZ reporter gene expression in response to a broad range of DNA-damaging agents and agents that interfere with DNA synthesis. All 11 tested known carcinogenic and genotoxic agents, ranging from DNA alkylating agents, oxidative chemicals and radiations, were able to induce RNR3-lacZ expression at a sublethal dose. In particular, a potent colon carcinogen, 1,2-dimethyl hydrazine (SDMH), was not detected as a mutagen by a standard Ames test, but was able to induce RNR3-lacZ expression. In contrast, both non-mutagenic and non-genotoxic chemicals tested were unable to induce RNR3-lacZ expression. We have compared three yeast DNA damage-inducible genes for their sensitivity and inducibility, and found that RNR3 is more sensitive than RNR2 and MAG1. The effects of damage dose, post-treatment incubation time and cell growth stage on RNR3-lacZ expression have also been determined and optimized. In order to create a stable and user-friendly testing system, we integrated the RNR3-lacZ cassette into the yeast genome and demonstrated that its inducibility is indistinguishable from that of plasmid-based studies.

[Study of ABO blood group secretor type alpha(1,2)-fucosyltransferase gene polymorphism in Chinese].

Secretor gene (FUT2)-encoded secretor type alpha(1,2)-fucosyltransferase (Se enzyme) that regulates expression and secretion of ABO(H) antigens in epithelial cell of glands and body fluids. It has the extensive polymorphism and race specificity. In the present study, we investigated the distribution of the fusion gene of the FUT2 locus in 191 Manchu individuals from Liaoning Province and 208 Mongolian individuals from Inner Mongolia, and analyzed the polymorphism of the FUT2 gene in 90 unrelated Han Chinese from Shandong Province and 90 Mongolian from Inner Mongolia, respectively. The fusion gene was not found in the two investigated ethnic groups. The frequencies of G849A nonsense mutation in Shandong Han Chinese and Mongolian of Inner Mongolia individuals were the same, 0.0055.

Methylglyoxal mediates adipocyte proliferation by increasing phosphorylation of Akt1.

Methylglyoxal (MG) is a highly reactive metabolite physiologically presented in all biological systems. The effects of MG on diabetes and hypertension have been long recognized. In the present study, we investigated the potential role of MG in obesity, one of the most important factors to cause metabolic syndrome. An increased MG accumulation was observed in the adipose tissue of obese Zucker rats. Cell proliferation assay showed that 5-20 µM of MG stimulated the proliferation of 3T3-L1 cells. Further study suggested that accumulated-MG stimulated the phosphorylation of Akt1 and its targets including p21 and p27. The activated Akt1 then increased the activity of CDK2 and accelerated the cell cycle progression of 3T3-L1 cells. The effects of MG were efficiently reversed by advanced glycation end product (AGE) breaker alagebrium and Akt inhibitor SH-6. In summary, our study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulation of Akt signaling pathway and this mechanism might offer a new approach to explain the development of obesity.

Zhenqing recipe alleviates diabetic nephropathy in experimental type 2 diabetic rats through suppression of SREBP-1c.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhenqing Recipe (ZQR), a Chinese herbal prescription, is used to improve renal function of patients with diabetic nephropathy. In current research, the therapeutic effects of ZQR on type 2 diabetic nephropathy and the underlying molecular mechanisms were explored. MATERIALS AND METHODS: Animal model with diabetic nephropathy was developed by high fat/sucrose diet feeding plus streptozotocin injection for 4 weeks. The diabetic rats were then orally administered with ZQR extract at the dose of 4 g/kg, 8 g/kg body weight/day for 8 weeks. RESULTS: Serum glucose, triglyceride and total cholesterol in untreated diabetic rats were significantly higher than that of normal control rats. ZQR treatment not only reduced serum glucose level in diabetic rats, but also decreased serum triglyceride and total cholesterol in a dose-dependent manner. Urinary albumin excretion rate, serum urea and creatinine were significantly decreased in ZQR groups compared with untreated diabetic group. Histopathological study of kidney samples showed that extracellular mesangial matrix expansion in diabetic rats was suppressed by ZQR treatment. Both mRNA and protein levels of sterol regulatory element binding-protein-1c (SREBP-1c), and its target genes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in renal cortex were significantly decreased in ZQR treated rats compared to untreated diabetic rats. Consequently, renal triglyceride was significantly reduced in ZQR groups. Furthermore, ZQR significantly inhibited the overexpression of transforming growth factor-ß1 and fibronectin in the renal cortex of diabetic rats. CONCLUSIONS: Oral treatment of ZQR improved diabetic nephropathy by inhibiting the overexpression of SREBP-1c and its target genes including ACC and FAS in experimental type 2 diabetic rats.

Zhenqing recipe improves glucose metabolism and insulin sensitivity by repressing hepatic FOXO1 in type 2 diabetic rats.

Forkhead box O1 (FOXO1) plays an important role in glucose metabolism at the gene transcription level. Increased FOXO1 activity results in hyperglycemia by promoting the expression of gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), and inhibiting glucokinase (GK). This study evaluates the effect of Zhenqing Recipe (ZQR), a Chinese herbal medicine, on hyperglycemia and its molecular mechanisms. Type 2 diabetic rats, developed by high-fat diet combined with low-dose STZ injections, were randomly divided into untreated diabetic, ZQR and metformin group. Normal rats served as control. After an eight-week treatment, fasting blood glucose was significantly decreased and insulin sensitivity index was obviously increased in the ZQR group. ZQR also improved the oral glucose tolerance. Compared with the control group, the mRNA levels of PEPCK and G6Pase were significantly elevated, while GK mRNA expression was decreased in the liver of untreated diabetic rats. ZQR significantly reduced the mRNA levels of PEPCK and G6Pase, and increased GK mRNA expression. The hepatic mRNA and protein expression of FOXO1 in the untreated diabetic group was markedly increased compared to controls. The administration of ZQR significantly decreased the mRNA and protein levels of hepatic FOXO1. The data suggest that ZQR improves glucose metabolism and insulin sensitivity, which is accompanied with regulating mRNA expression of GK and gluconeogenic genes. This anti-diabetic effect of ZQR is due to its ability to repress hepatic FOXO1 at the mRNA and protein level.

Accumulation of endogenous methylglyoxal impaired insulin signaling in adipose tissue of fructose-fed rats.

Increased accumulation of methylglyoxal (MG) has been linked to different insulin resistance states including diabetes and hypertension. In this study, the effects of MG on insulin signaling pathway were investigated. Following 9 weeks of fructose treatment, an insulin resistance state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, high blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. More importantly, we observed a close correlation between the development of insulin resistance and elevated MG level in serum and adipose tissue. Both insulin resistance state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC). When 3T3-L1 adipocytes were treated directly with MG, the impaired insulin signaling was also observed, indicated by decreased insulin-induced insulin-receptor substrate-1 (IRS-1) tyrosine phosphorylation and the decreased kinase activity of phosphatidylinositol (PI) 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In conclusion, the increase in endogenous MG accumulation impairs insulin-signaling pathway and decreases insulin-stimulated glucose uptake in adipose tissue, which may contribute to the development of insulin resistance.

Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.

H2S is an important gasotransmitter, generated in mammalian cells from L-cysteine metabolism. As it stimulates K(ATP) channels in vascular smooth muscle cells, H2S may also function as an endogenous opener of K(ATP) channels in INS-1E cells, an insulin-secreting cell line. In the present study, K(ATP) channel currents in INS-1E cells were recorded using the whole-cell and single-channel recording configurations of the patch-clamp technique. K(ATP) channels in INS-1E cells have a single-channel conductance of 78 pS. These channels were activated by diazoxide and inhibited by gliclazide. ATP (3 mm) in the pipette solution inhibited K(ATP) channels in INS-1E cells. Significant amount of H2S was produced from INS-1E cells in which the expression of cystathinonie gamma-lyase (CSE) was confirmed. After INS-1E cells were transfected with CSE-targeted short interfering RNA (CSE-siRNA) or treated with DL-propargylglycine (PPG; 1-5 mm) to inhibit CSE, endogenous production of H2S was abolished. Increase in extracellular glucose concentration significantly decreased endogenous production of H2S in INS-1E cells, and increased insulin secretion. After transfection of INS-1E cells with adenovirus containing the CSE gene (Ad-CSE) to overexpress CSE, high glucose-stimulated insulin secretion was virtually abolished. Basal K(ATP) channel currents were significantly reduced after incubating INS-1E cells with a high glucose concentration (16 mm) or lowering endogenous H2S level by CSE-siRNA transfection. Under these conditions, exogenously applied H2S significantly increased whole-cell K(ATP) channel currents at concentrations equal to or lower than 100 microm. H2S (100 microm) markedly increased open probability by more than 2-fold of single K(ATP) channels (inside-out recording) in native INS-1E cells (n = 4, P < 0.05). Single-channel conductance and ATP sensitivity of K(ATP) channels were not changed by H2S. In conclusion, endogenous H2S production from INS-1E cells varies with in vivo conditions, which significantly affects insulin secretion from INS-1E cells. H2S stimulates K(ATP) channels in INS-1E cells, independent of activation of cytosolic second messengers, which may underlie H2S-inhibited insulin secretion from these cells. Interaction among H2S, glucose and the K(ATP) channel may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic beta-cells.

Y-chromosomal DNA haplogroups and their implications for the dual origins of the Koreans.

We have analyzed eight Y-chromosomal binary markers (YAP, RPS4Y(711), M9, M175, LINE1, SRY(+465), 47z, and M95) and three Y-STR markers (DYS390, DYS391, and DYS393) in 738 males from 11 ethnic groups in east Asia in order to study the male lineage history of Korea. Haplogroup DE-YAP was found at a high frequency only in Japan but was also present at low frequencies in northeast Asia, including 2.5% in Korea, suggesting a northern origin for these chromosomes. Haplogroup C-RPS4Y(711) was present in Korea and Manchuria at moderate frequencies: higher than in populations from southeast Asia, but lower than those in the northeast, which may imply a northern Asian expansion of these lineages, perhaps from Mongolia or Siberia. The major Y-chromosomal expansions in east Asia were those of haplogroup O-M175 (and its sublineages). This haplogroup is likely to have originated in southern east Asia and subsequently expanded to all of east Asia. The moderate frequency of one sublineage in the Koreans, haplogroup O-LINE1 (12.5%), could be a result of interaction with Chinese populations. The age of another sublineage, haplogroup O-SRY(+465), and Y-STR haplotype diversity provide evidence for relatively recent male migration, originally from China, through Korea into Japan. In conclusion, the distribution pattern of Y-chromosomal haplogroups reveals the complex origin of the Koreans, resulting from genetic contributions involving the northern Asian settlement and range expansions mostly from southern-to-northern China.