Plasma vitamin C levels in ESRD patients and occurrence of hypochromic erythrocytes.

INTRODUCTION: The achievement of erythropoiesis in hemodialysis (HD) patients is typically managed with erythropoiesis-stimulating-agents (ESA's) and intravenous iron (IV-iron). Using this treatment strategy, HD patients frequently show an elevated fraction of red blood cells (RBC) with hemoglobin (Hb) content per cell that is below the normal range, called hypochromic RBC. The low Hb content per RBC is the result of the clinical challenge of providing sufficient iron content to the bone marrow during erythropoiesis. Vitamin C supplements have been used to increase Hb levels in HD patients with refractory anemia, which supports the hypothesis that vitamin C mobilizes iron needed for Hb synthesis. METHODS: We conducted a cross-sectional survey in 149 prevalent HD patients of the percent hypochromic RBC, defined as RBC with Hb < 300 ng/uL of packed RBC, in relation to plasma vitamin C levels. We also measured high-sensitivity CRP, (hs-CRP), iron, and ferritin levels. and calculated ESA dose. FINDINGS: High plasma levels of vitamin C were negatively associated with hypochromic RBC (P < 0.003), and high ESA doses were positively associated (P < 0.001). There was no significant association of hs-CRP with percent hypochromic RBC. DISCUSSION: This finding supports the hypothesis that vitamin C mobilizes iron stores, improves iron delivery to the bone marrow, and increase the fraction of RBC with normal Hb content. Further research is warranted on development of protocols for safe and effective use of supplemental vitamin C for management of renal anemia.

KPNBeta1 promotes palmitate-induced insulin resistance via NF-kappaB signaling in hepatocytes

It has been intensively studied that inflammation contributes to the insulin resistance development in obesity-induced type 2 diabetes mellitus (T2DM). In this study, we assessed the effect of karyopherin Beta1 (KPNBeta1) in hepatic insulin resistance and the underlying mechanisms using high-fat diet (HFD) fed mice and palmitate (PA)-stimulated hepatocytes (HepG2). KPNBeta1 expression is increased in the HFD fed mice liver. PA upregulated KPNBeta1 expression in HepG2 cells in a time-dependent manner. PA also increased pro-inflammatory cytokines expression, including tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and interleukin 1Beta (IL-1Beta). KPNBeta1 knockdown reversed PA-induced pro-inflammatory cytokines expression and insulin-stimulated glucose uptake in HepG2 cells. In addition, KPNBeta1 knockdown reduced intracellular lipid accumulation. Mechanistically, KPNBeta1 transports nuclear factor kB (NF-kappaB) p65 from the cytoplasm to the nucleus to increase pro-inflammatory genes expression. In summary, KPNBeta1 acts as a positive regulator in the NF-kappaB pathway to enhance palmitate-induced inflammation response and insulin resistance in HepG2 cells

TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway.

High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3ß was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3ß in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3ß. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway.

KPNß1 promotes palmitate-induced insulin resistance via NF-κB signaling in hepatocytes.

It has been intensively studied that inflammation contributes to the insulin resistance development in obesity-induced type 2 diabetes mellitus (T2DM). In this study, we assessed the effect of karyopherin ß1 (KPNß1) in hepatic insulin resistance and the underlying mechanisms using high-fat diet (HFD) fed mice and palmitate (PA)-stimulated hepatocytes (HepG2). KPNß1 expression is increased in the HFD fed mice liver. PA upregulated KPNß1 expression in HepG2 cells in a time-dependent manner. PA also increased pro-inflammatory cytokines expression, including tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 1ß (IL-1ß). KPNß1 knockdown reversed PA-induced pro-inflammatory cytokines expression and insulin-stimulated glucose uptake in HepG2 cells. In addition, KPNß1 knockdown reduced intracellular lipid accumulation. Mechanistically, KPNß1 transports nuclear factor kB (NF-κB) p65 from the cytoplasm to the nucleus to increase pro-inflammatory genes expression. In summary, KPNß1 acts as a positive regulator in the NF-κB pathway to enhance palmitate-induced inflammation response and insulin resistance in HepG2 cells.

TAB3 involves in hepatic insulin resistance through activation of MAPK pathway.

Insulin resistance is often accompanied by chronic inflammatory responses. The mitogen-activated protein kinase (MAPK) pathway is rapidly activated in response to many inflammatory cytokines. But the functional role of MAPKs in palmitate-induced insulin resistance has yet to be clarified. In this study, we found that transforming growth factor ß-activated kinase binding protein-3 (TAB3) was up-regulated in insulin resistance. Considering the relationship between transforming growth factor ß-activated kinase (TAK1) and MAPK pathway, we assumed TAB3 involved in insulin resistance through activation of MAPK pathway. To certify this hypothesis, we knocked down TAB3 in palmitate treated HepG2 cells and detected subsequent biological responses. Importantly, TAB3 siRNA directly reversed insulin sensitivity by improving insulin signal transduction. Moreover, silencing of TAB3 could facilitate hepatic glucose uptake, reverse gluconeogenesis and improve ectopic fat accumulation. Meanwhile, we found that the positive effect of knocking down TAB3 was more significant when insulin resistance occurred. All these results indicate that TAB3 acts as a negative regulator in insulin resistance through activation of MAPK pathway.

The Role of PTP1B O-GlcNAcylation in Hepatic Insulin Resistance.

Protein tyrosine phosphatase 1B (PTP1B), which can directly dephosphorylate both the insulin receptor and insulin receptor substrate 1 (IRS-1), thereby terminating insulin signaling, reportedly plays an important role in insulin resistance. Accumulating evidence has demonstrated that O-GlcNAc modification regulates functions of several important components of insulin signal pathway. In this study, we identified that PTP1B is modified by O-GlcNAcylation at three O-GlcNAc sites (Ser104, Ser201, and Ser386). Palmitate acid (PA) impaired the insulin signaling, indicated by decreased phosphorylation of both serine/threonine-protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3ß) following insulin administration, and upregulated PTP1B O-GlcNAcylation in HepG2 cells. Compared with the wild-type, intervention PTP1B O-GlcNAcylation by site-directed gene mutation inhibited PTP1B phosphatase activity, resulted in a higher level of phosphorylated Akt and GSK3ß, recovered insulin sensitivity, and improved lipid deposition in HepG2 cells. Taken together, our research showed that O-GlcNAcylation of PTP1B can influence insulin signal transduction by modulating its own phosphatase activity, which participates in the process of hepatic insulin resistance.

PRDX1 is involved in palmitate induced insulin resistance via regulating the activity of p38MAPK in HepG2 cells.

Studies have identified that type 2 diabetes mellitus (T2DM) patients displayed higher levels of plasma peroxiredoxin1(PRDX1) than non-diabetics. However, the impact of PRDX1 on insulin resistance and the underlying mechanism remains totally unknown. Here, we investigated the influence of PRDX1 on hepatic insulin resistance. We showed that the protein and mRNA levels of PRDX1 were significantly elevated under insulin-resistant conditions. In addition, we showed that interference of PRDX1 ameliorated palmitate-induced insulin resistance in HepG2 cells, which was indicated by elevated phosphorylation of protein kinase B (AKT) and of glycogen synthase kinase-3 (GSK3ß). Furthermore, the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), two key gluconeogenic enzymes, were down-regulated following PRDX1 depletion. Accordingly, glucose uptake was suppressed in PRDX1-interferred HepG2 cells. In addition, Over-expression of PRDX1 enhanced PA-induced insulin resistance in HepG2 cells. Moreover, we found that knocking down PRDX1 improves insulin sensitivity and decreased the activation of p38 mitogen-activated protein kinase (p38MAPK). Our results demonstrate that PRDX1 can induce hepatic insulin resistance by activating p38MAPK signaling and identifies potential targets for new treatments.

PCBP2 regulates hepatic insulin sensitivity via HIF-1α and STAT3 pathway in HepG2 cells.

Elevated free fatty acids (FFAs) are fundamental to the pathogenesis of hepatic insulin resistance. However, the molecular mechanisms of insulin resistance remain not completely understood. Transcriptional dysregulation, post-transcriptional modifications and protein degradation contribute to the pathogenesis of insulin resistance. Poly(C) binding proteins (PCBPs) are RNA-binding proteins that are involved in post-transcriptional control pathways. However, there are little studies about the roles of PCBPs in insulin resistance. PCBP2 is the member of the RNA-binding proteins and is thought to participate in regulating hypoxia inducible factor-1 (HIF-1α) and signal transducers and activators of transcription (STAT) pathway which are involved in regulating insulin signaling pathway. Here, we investigated the influence of PCBP2 on hepatic insulin resistance. We showed that the protein and mRNA levels of PCBP2 were down-regulated under insulin-resistant conditions. In addition, we showed that over-expression of PCBP2 ameliorates palmitate (PA)-induced insulin resistance, which was indicated by elevated phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3ß (GSK3ß). We also found that over-expression of PCBP2 inhibits HIF1α and STAT3 pathway. Furthermore, glucose uptake was found to display a similar tendency with the phosphorylation of Akt. The expressions of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), two key gluconeogenic enzymes, were down-regulated following Over-expression of PCBP2. Accordingly, PA-induced intracellular lipid accumulation was suppressed in over-expression of PCBP2 HepG2 cells. In addition, we found that over-expression of PCBP2 inhibits HIF1α and STAT3 pathway. Our results demonstrate that PCBP2 was involved in hepatic insulin sensitivity might via HIF-1α and STAT3 pathway in HepG2 cells.

TRAM1 protect HepG2 cells from palmitate induced insulin resistance through ER stress-JNK pathway.

Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. With high-fat feeding (HFD), FFAs can activate chronic endoplasmic reticulum (ER) stress in target tissues, initiating negative crosstalk between FFAs and insulin signaling. However, the molecular link between insulin resistance and ER stress remains to be identified. We here reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, was involved in the onset of insulin resistance in hepatocytes. TRAM1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAM1 led to hyperactivation of CHOP and GRP78, and the activation of downstream JNK pathway. Given the fact that the activation of ER stress played a facilitating role in insulin resistance, the phosphorylation of Akt and GSK-3ß was also analyzed. We found that depletion of TRAM1 markedly attenuated the phosphorylation of Akt and GSK-3ß in the cells. Moreover, application with JNK inhibitor SP600125 reversed the effect of TRAM1 interference on Akt phosphorylation. The accumulation of lipid droplets and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt. Glucose uptake assay indicated that knocking down TRAM1 augmented PA-induced down-regulation of glucose uptake, and inhibition of JNK using SP600125 could block the effect of TRAM1 on glucose uptake. These data implicated that TRAM1 might protect HepG2 cells against PA-induced insulin resistance through alleviating ER stress.

Gibberellin biosynthetic deficiency is responsible for maize dominant Dwarf11 (D11) mutant phenotype: physiological and transcriptomic evidence.

Dwarf stature is introduced to improve lodging resistance and harvest index in crop production. In many crops including maize, mining and application of novel dwarf genes are urgent to overcome genetic bottleneck and vulnerability during breeding improvement. Here we report the characterization and expression profiling analysis of a newly identified maize dwarf mutant Dwarf11 (D11). The D11 displays severely developmental abnormalities and is controlled by a dominant Mendelian factor. The D11 seedlings responds to both GA3 and paclobutrazol (PAC) application, suggesting that dwarf phenotype of D11 is caused by GA biosynthesis instead of GA signaling deficiency. In contrast, two well-characterized maize dominant dwarf plants D8 and D9 are all insensitive to exogenous GA3 stimulation. Additionally, sequence variation of D8 and D9 genes was not identified in the D11 mutant. Microarray and qRT-PCR analysis results demonstrated that transcripts encoding GA biosynthetic and catabolic enzymes ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), and GA 2-oxidase (GA2ox) are up-regulated in D11. Our results lay a foundation for the following D11 gene cloning and functional characterization. Moreover, results presented here may aid in crops molecular improvement and breeding, especially breeding of crops with plant height ideotypes.

Systematic analysis of plant-specific B3 domain-containing proteins based on the genome resources of 11 sequenced species.

B3 domain-containing proteins constitute a large transcription factor superfamily. The plant-specific B3 superfamily consists of four family members, i.e., LAV (LEC2 [LEAFY COTYLEDON 2]/ABI3 [ABSCISIC ACID INSENSITIVE 3] − VAL [VP1/ABI3-LIKE]), RAV (RELATED to ABI3/VP1), ARF (AUXIN RESPONSE FACTOR) and REM (REPRODUCTIVE MERISTEM) families. The B3 superfamily plays a central role in plant life, from embryogenesis to seed maturation and dormancy. In previous research, we have characterized ARF family, member of the B3 superfamily in silico (Wang et al., Mol Biol Rep, 2011, doi:10.1007/s11033-011-0991-z). In this study, we systematically analyzed the diversity, phylogeny and evolution of B3 domain-containing proteins based on genomic resources of 11 sequenced species. A total of 865 B3 domain-containing genes were identified from 11 sequenced species through an iterative strategy. The number of B3 domain-containing genes varies not only between species but between gene families. B3 domain-containing genes are unevenly distributed in chromosomes and tend to cluster in the genome. Numerous combinations of B3 domains and their partner domains contribute to the sequences and structural diversification of the B3 superfamiy. Phylogenetic results showed that moss VAL proteins are related to LEC2/ABI3 instead of VAL proteins from higher plants. Lineage-specific expansion of ARF and REM proteins was observed. The REM family is the most diversified member among the B3 superfamily and experiences a rapid divergence during selective sweep. Based on structural and phylogenetic analysis results, two possible evolutional modes of the B3 superfamily were presented. Results presented here provide a resource for further characterization of the B3 superfamily.

Hemoglobin and plasma vitamin C levels in patients on peritoneal dialysis.

OBJECTIVE: To determine the contribution of vitamin C (Vit C) status in relation to hemoglobin (Hb) levels in patients on long-term peritoneal dialysis (PD). METHODS: 56 stable PD patients were evaluated in a cross-sectional survey. Plasma samples were collected for Vit C (analyzed by HPLC with electrochemical detection) and high-sensitivity C-reactive protein (hs-CRP) determinations. Clinical records were reviewed for Hb, transferrin saturation (TSAT), ferritin, erythropoietin (EPO) dose, and other clinical parameters. Dietary Vit C intake was evaluated by patient survey and from patient records. Total Vit C removed during PD treatment was measured in 24-hour dialysate collections. RESULTS: Patients showed a highly skewed distribution of plasma Vit C levels, with 40% of patients below normal plasma Vit C levels (<30 µmol/L) and 9% at higher than normal levels (>80 µmol/L). Higher plasma Vit C levels were associated with higher Hb levels (Pearson r = 0.33, p < 0.004). No direct connection between Vit C levels and reported dietary intake could be established. In stepwise multiple regression, plasma Vit C remained significantly associated with Hb (p = 0.017) but there was no significant association with other variables (dialysis vintage, age, ferritin, TSAT, hs-CRP, residual renal function, and EPO dose). In 9 patients that were evaluated for Vit C in dialysate, plasma Vit C was positively associated (Spearman r = 0.85, p = 0.01) with the amount of Vit C removed during dialysis treatment. CONCLUSIONS: These data indicate that plasma Vit C is positively associated with higher Hb level. Vit C status could play a major role in helping PD patients to utilize iron for erythropoiesis and achieve a better Hb response during anemia management.

Chemical reactions of vitamin C with intravenous-iron formulations.

BACKGROUND: Intravenous (IV) iron is widely prescribed for patients on haemodialysis, to replace iron losses during treatment. It releases labile iron, which can induce oxidation of vitamin C and trigger oxidant damage. We examined the stability of vitamin C in the presence of IV iron compounds. We further examined in the ability of vitamin C to release iron from these compounds. METHODS: Vitamin C was measured by high-performance liquid chromatography with electrochemical detection. Iron release from iron sucrose (FeSuc) and ferric gluconate (FeGlu) was determined with the ferrozine method. RESULTS: Vitamin C, in human plasma or fetal calf serum, was oxidized in this order of reactivity: FeSuc > FeGlu > blank reaction. FeSuc and FeGlu also oxidized vitamin C when added to freshly obtained whole human blood. During a 4 h incubation in buffer, vitamin C stimulated the release of 60% of the iron content of FeSuc at p 4, with lesser amounts at pH 3, 5 and 6, and 5% release at pH 7. Vitamin C also triggered the release of iron from FeGlu, but less release was observed than with FeSuc. Using ferrozine reagent, no iron release was detected to heparinized human plasma, following addition of 500 microM concentrations of iron compounds. CONCLUSION: Each IV-iron compound can oxidize substantial amounts of vitamin C when added to plasma or whole blood. The interaction of vitamin C is accompanied by release of iron from the particle at mildly acidic pH, which may explain the ability of high-dose vitamin C to mobilize iron from storage sites for erythropoiesis.