Metabolomic profiling of embryo culture media in patients with repeated implantation failure during assisted reproductive technology cycles.

OBJECTIVE: This study investigated the metabolic status of the spent culture media from embryos of patients with repeated implantation failure (RIF) undergoing in vitro fertilization-intracytoplasmic sperm injection cycles in comparison with the embryos from healthy fertile women. METHODS: Metabolite levels in spent culture media were assessed and compared between embryos from RIF patients (n=35) and oocyte donors as controls (n=15). Protein levels of insulin-like growth factor 1 (IGF-1) were determined using Western blotting. Concentrations of glucose, pyruvate, and lactate were measured using spectrophotometry. Ionic colorimetric assay kits were utilized to analyze the concentrations of sodium, chloride, calcium, and magnesium ions. High-performance liquid chromatography was employed to measure the concentrations of glutamic acid, aspartic acid, methionine, phenylalanine, and histidine. RESULTS: Glucose consumption and lactate secretion were higher in the control group than in the RIF group. The magnesium concentration was significantly higher in the control group than in the RIF group, but glutamic acid and aspartic acid concentrations were lower in the control group than in the RIF patients (p<0.05). The levels of IGF-1, sodium, calcium, chloride, methionine, histidine, and phenylalanine did not show statistically significant differences between the two groups. CONCLUSION: The metabolic profile of the culture medium of the embryos in the RIF group differed from that of the control group. These findings suggest potential factors that may affect implantation capacity in RIF patients and provide a new perspective on embryo selection.

SIRT1 activation attenuates palmitate induced apoptosis in C2C12 muscle cells.

BACKGROUND: Type 2 diabetes is characterized by insulin resistance, which manifests mainly in skeletal muscles. SIRT1 has been found to play a role in the insulin signaling pathway. However, the molecular underpinnings of SIRT1's function in palmitate fatty acid-induced apoptosis still need to be better understood. METHODS: In this research, skeletal muscle cells are treated with palmitate to be insulin resistant. It is approached that SIRT1 is downregulated in C2C12 muscle cells during palmitate-induced apoptosis and that activating SIRT1 mitigates this effect. RESULTS: Based on these findings, palmitate-induced apoptosis suppressed mitochondrial biogenesis by lowering PGC-1 expression, while SIRT1 overexpression boosted. The SIRT1 inhibitor sirtinol, on the other hand, decreased mitochondrial biogenesis under the same conditions. This research also shows that ROS levels rise in the conditions necessary for apoptosis induction by palmitate, and ROS inhibitors can mitigate this effect. This work demonstrated that lowering ROS levels by boosting SIRT1 expression inhibited apoptotic induction in skeletal muscle cells. CONCLUSION: This study's findings suggested that SIRT1 can improve insulin resistance in type 2 diabetes by slowing the rate of lipo-apoptosis and boosting mitochondrial biogenesis, among other benefits.

Molecular mechanism of the anti-diabetic activity of an identified oligosaccharide from Rosa canina.

BACKGROUND AND PURPOSE: Because of the high prevalence, diabetes is considered a global health threat. Hence, the need for effective, cheap, and comfortable therapies are highly felt. In previous study, a novel oligosaccharide with strong anti-diabetic activity in the crude extract of Rosa canina fruits, from the rosacea family, was identified. The present study was designed to ensure its efficacy using in vivo and in vitro studies. EXPERIMENTAL APPROACH: Crude extract and its purified oligosaccharide were prepared from corresponding herb. Adult male Wistar rats were randomly divided into four groups of 10 each, as follows: group 1, healthy control rats given only sterile normal saline; group 2, diabetic control rats received sterile normal saline; group 3, diabetic rats treated with crude extract of Rosa canina (40% w/v) by oral gavage for 8 weeks; group 4, diabetic rats treated with purified oligosaccharide of Rosa canina (2 mg/kg) by oral gavage for 8 weeks. After treatment, body weight, fasting blood glucose, serum insulin levels and islet beta-cell repair and proliferation were investigated. The possible cytoprotective action of oligosaccharide was evaluated in vitro. The effect of oligosaccharide on apoptosis and insulin secretion in cell culture media were examined. Real-time PCR was used to determine the expression level of some glucose metabolism-related regulator genes. FINDINGS / RESULTS: In the animal model of diabetes, the insulin levels were increased significantly due to the regeneration of beta-cells in the islands of langerhans by the purified oligosaccharide. In vitro cell apoptosis examination showed that high concentration of oligosaccharide increased cell death, while at low concentration protected cells from streptozotocin-induced apoptosis. Molecular study showed that the expression of Ins1 and Pdx1 insulin production genes were increased, leading to increased expression of insulin-dependent genes such as Gck and Ptp1b. On the other hand, the expression of the Slc2a2 gene, which is related to the glucose transporter 2, was significantly reduced due to insulin concentrations. CONCLUSION AND IMPLICATIONS: The purified oligosaccharide from Rosa canina was a reliable anti-diabetic agent, which acted by increasing insulin production in beta-cells of the islands of Langerhans.

Investigating the mechanisms behind extensive death in human cancer cells following nanoparticle assisted photo-thermo-radiotherapy.

We have recently reported the synthesis and characterization of gold-coated iron oxide nanoparticle and demonstrated such a nanoparticle (Au@Fe2O3 NP) was able to significantly enhance the lethal effects of photo-thermo-radiotherapy. The purpose of this study was to determine the mechanisms behind such an enhancement by investigating the changes induced in cancer cell viability, proliferation, and morphology as well as monitoring the alteration of some genes which play important role in the process of cell death. Using MTT assay and transmission electron microscopy (TEM), the KB cells viability and morphology were assessed after treating with various combinations of NPs, photothermal therapy (PTT), and radiotherapy (RT). Clonogenic assay was used to assess the proliferation ability of treated KB cells. Nanoparticle internalization into the cells was investigated by TEM and inductively coupled plasma (ICP). During the treatment procedures, temperature changes were monitored using an IR-camera. Furthermore, the changes occurred in Bax, BCL2 and HSP70 genes expression level were measured using real-time PCR. The results showed that combination of NP, PTT, and RT caused more cell death compared to PTT or RT alone. Following such a combination therapy, massive cell injury was detected. We also observed an extensive increase in Bax/Bcl2 ratio and HSP70 expression for the KB cells treated by combination therapy procedure. Our results showed that massive cell injury and apoptosis induction are the main reasons of extensive cell death observed in cancer cells when a nanoparticle assisted photo-thermo-radiotherapy procedure is applied.

Co-administration of human adipose-derived stem cells and low-level laser to alleviate neuropathic pain after experimental spinal cord injury.

BACKGROUND: Evidence has suggested that human adipose-derived stem cells (hADSCs) and low-level laser has neuroprotective effects on spinal cord injury (SCI). Therefore, the combined effect of the hADSCs and laser on neuregeneration and neuropathic pain after SCI was investigated. METHODS: Forty-eight adult male Wistar rats with 200-250 g weight were used. Thirty minutes after compression, injury with laser was irritated, and 1 week following SCI, about 1 × 106 cells were transplanted into the spinal cord. Motor function and neuropathic pain were assessed weekly. Molecular and histological studies were done at the end of the fourth week. RESULTS: The combined application of hADSCs and laser has significantly improved motor function recovery (p = 0.0001), hyperalgesia (p < 0.05), and allodynia (p < 0.05). GDNF mRNA expression was significantly increased in hADSCs and laser+hADSC-treated animals (p < 0.001). Finally, co-administration of hADSCs and laser has enhanced the number of axons around cavity more than other treatments (p < 0.001). CONCLUSIONS: The results showed that the combination of laser and ADSCs could significantly improve the motor function and alleviate SCI-induced allodynia and hyperalgesia. Therefore, using a combination of laser and hADSCs in future experimental and translational clinical studies is suggested.

Genetic Analysis of 13 Iranian Families With Leukocyte Adhesion Deficiency Type 1.

BACKGROUND AND AIM: Leukocyte adhesion deficiency type 1 is a rare, autosomal recessive disorder that results from mutations in the ITGB2 gene. This gene encodes the CD18 subunit of ß2 integrin leukocyte adhesion cell molecules. Leukocyte adhesion deficiency type 1 is characterized by recurrent bacterial infections, impaired wound healing, inadequate pus formation, and delayed separation of the umbilical cord. MATERIALS AND METHODS: Blood samples were taken from 13 patients after written consent had been obtained. Genomic DNA was extracted, and ITGB2 exons and exon-intron boundaries were amplified by polymerase chain reaction. The products were examined by Sanger sequencing. RESULTS: In this study, 8 different previously reported mutations (intron7+1G>A, c.715G>A, c.1777 C>T, c.843del C, c.1768T>C, c.1821C>A, Intron7+1G>A, c.1885G>A) and 2 novel mutations (c.1821C>A; p.Tyr607Ter and c.1822C>T; p.Gln608Ter) were found. CONCLUSIONS: c.1821C>A (p.Tyr607Ter) and c.1822C>T (p.Gln608Ter) mutations should be included in the panel of carrier detection and prenatal diagnosis.

Diet and cancer prevention: Dietary compounds, dietary MicroRNAs, and dietary exosomes.

Cancer is one of main health public problems worldwide. Several factors are involved in beginning and development of cancer. Genetic and internal/external environmental factors can be as important agents that effect on emerging and development of several cancers. Diet and nutrition may be as one of important factors in prevention or treatment of various cancers. A large number studies indicated that suitable dietary patterns may help to cancer prevention or could inhibit development of tumor in cancer patients. Moreover, a large numbers studies indicated that a variety of dietary compounds such as curcumin, green tea, folat, selenium, and soy isoflavones show a wide range anti-cancer properties. It has been showed that these compounds via targeting a sequence of cellular and molecular pathways could be used as suitable options for cancer chemoprevention and cancer therapy. Recently, dietary microRNAs and exosomes have been emerged as attractive players in cancer prevention and cancer therapy. These molecules could change behavior of cancer cells via targeting various cellular and molecular pathways involved in cancer pathogenesis. Hence, the utilization of dietary compounds which are associated with powerful molecules such as microRNAs and exosomes and put them in dietary patterns could contribute to prevention or treatment of various cancers. Here, we summarized various studies that assessed effect of dietary patterns on cancer prevention shortly. Moreover, we highlighted the utilization of dietary compounds, dietary microRNAs, and dietary exosomes and their cellular and molecular pathways in cancer chemoprevention.

Molecular aspects of diabetes mellitus: Resistin, microRNA, and exosome.

Diabetes mellitus (DM) is known as one of important common endocrine disorders which could due to deregulation of a variety of cellular and molecular pathways. A large numbers studies indicated that various pathogenesis events including mutation, serin phosphorylation, and increasing/decreasing expression of many genes could contribute to initiation and progression of DM. Insulin resistance is one of important factors which could play critical roles in DM pathogenesis. It has been showed that insulin resistance via targeting a sequence of cellular and molecular pathways (eg, PI3 kinases, PPARγ co-activator-1, microRNAs, serine/threonine kinase Akt, and serin phosphorylation) could induce DM. Among of various factors involved in DM pathogenesis, microRNAs, and exosomes have been emerged as effective factors in initiation and progression of DM. A variety of studies indicated that deregulation of these molecules could change behavior of various types of cells and contribute to progression of DM. Resistin is other main factor which is known as signal molecule involved in insulin resistance. Multiple lines evidence indicated that resistin exerts its effects via affecting on glucose metabolism, inhibition of fatty acid uptake and metabolism with affecting on a variety of targets such as CD36, fatty acid transport protein 1, Acetyl-CoA carboxylase, and AMP-activated protein kinase. Here, we summarized various molecular aspects are associated with DM particularly the molecular pathways involved in insulin resistance and resistin in DM. Moreover, we highlighted exosomes and microRNAs as effective players in initiation and progression of DM.

Association between Leptin G2548A and Leptin Receptor Q223R Polymorphisms with Type 2 Diabetes in an Iranian Population.

BACKGROUND: The leptin (LEP G2548A) and leptin receptor (LEPR Q223R) gene polymorphisms have been variably associated with type 2 diabetes (T2D) in different populations. In this study we hypothesized that these variants might be associated with T2D and related metabolic traits in an Iranian population. METHODS: The LEP G2548A and LEPR Q223R genotypes were determined by PCR-RFLP in 378 normoglycemic controls and 154 T2D patients. Bonferroni correction was applied for the correction of multiple testing. RESULTS: The A allele of the LEP G2548A polymorphism was more prevalent in females of the T2D group than the controls (p = 0.009). In a recessive model (GG+GA vs. AA), the frequency of the AA genotype was higher in female patients compared to normoglycemic subjects 134.9% vs. 19.3%, OR 2.60 (1.27-5.31), p = 0.0091. Multivariate logistic regression analysis also showed that the AA genotype of the LEP G2548A polymorphism is an independent risk factor for T2D in females. No significant association was found between the allele and genotype frequencies of the LEPR Q223R variant with T2D in female and male groups. In addition, no significant difference in anthropometrical and biochemical parameters was observed between the genotypes of LEP and LEPR variants in gender-specific groups in both non-diabetic and diabetic subjects. CONCLUSIONS: Our results suggest that the LEP G2548A polymorphisms might associate with T2D among Iranian female subjects, whereas the LEPR Q223R variant is not associated with T2D and its related metabolic traits in this population.

TNF-α knockdown alleviates palmitate-induced insulin resistance in C2C12 skeletal muscle cells.

Insulin resistance is a cardinal feature of Type 2 Diabetes (T2D), which accompanied by lipid accumulation and TNF-α overexpression in skeletal muscle. The role of TNF-α in palmitate-induced insulin resistance remained to be elucidated. Here, we assessed effects of TNF-α knockdown on the components of insulin signaling pathway (IRS-1 and Akt) in palmitate-induced insulin resistant C2C12 skeletal muscle cells. To reduce TNF-α expression, C2C12 cells were transduced with TNF-α-shRNA lentiviral particles. Afterwards, the protein expression of TNF-α, IRS-1, and Akt, as well as phosphorylation levels of IRS-1 and Akt were evaluated by western blot. We also measured insulin-stimulated glucose uptake in the presence and absence of palmitate. TNF-α protein expression in C2C12 cells significantly increased by treatment with 0.75 mM palmitate (P < 0.05). In TNF-α knockdown cells, the protein expression level of TNF-α was significantly decreased by almost 70% (P < 0.01) compared with the control cells. Our results also revealed that, in control cells, palmitate treatment significantly reduced the insulin-induced phosphorylations of IRS-1 (Tyr632) and Akt (Ser473) by 60% and 66% (P < 0.01), respectively. Interestingly, these phosphorylations, even in the presence of palmitate, were not significantly reduced in TNF-α knockdown cells with respect to the untreated control cells (P > 0.05). Furthermore, palmitate significantly reduced insulin-dependent glucose uptake in control cells, however, it was not able to reduce insulin-stimulated glucose uptake in TNF-α knockdown cells in comparison with the untreated control cells (P < 0.01). These findings indicated that TNF-α down-regulation maintains insulin sensitivity, even in the presence of palmitate, therefore, TNF-α inhibition could be a good strategy for the treatment of palmitate-induced insulin resistance.

Rosiglitazone, a PPARγ agonist, ameliorates palmitate-induced insulin resistance and apoptosis in skeletal muscle cells.

UNLABELLED: Palmitate induces insulin resistance and apoptosis in insulin target tissues. Rosiglitazone (RSG), a peroxisome proliferator-activated receptor γ (PPARγ) agonist, can activate both pro-apoptotic and anti-apoptotic pathways in different cells; however, its effect on palmitate-induced apoptosis in skeletal muscle cells remains to be elucidated. After differentiation of C2C12 cells, myotubes were treated with palmitate, RSG and GW9662 (PPARγ antagonist). MTT and terminal deoxynucleotide transferase dUTP nick end labelling (TUNEL) assays and caspase-3 activity were used to investigate the apoptosis. To study the underlying mechanism, glucose uptake, gene expression and protein levels were evaluated. A total of 0.75 mM palmitate reduced cell viability by 43% and increased TUNEL-positive cells and caspase-3 activity by 15-fold and 6.6-fold, respectively. RSG (10 µM) could markedly decrease the level of TUNEL-positive cells and caspase-3 activity in palmitate-treated cells. The protective effect of RSG on apoptosis was abrogated by GW9662. To investigate the molecular mechanism of this effect, gene expression and protein level of protein tyrosine phosphatase 1B (PTP1B) were evaluated. Palmitate and RSG individually increased the expression and protein level of PTP1B, whereas combined treatment (palmitate and RSG) were able to further increase the expression of PTP1B in C2C12 cells. We also evaluated the effect of RSG on palmitate-induced insulin resistance in muscle cells. RSG could significantly improve glucose uptake by 0.4-fold in myotubes treated with palmitate. Moreover, RSG could restore the phosphorylation of Akt in palmitate-treated cells. These data suggest that RSG protects skeletal muscle cells against palmitate-induced apoptosis and this effect appears to be mediated via the PPARγ-dependent and PTP1B-independent mechanisms. SIGNIFICANCE OF THE STUDY: Saturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis. Strategies for preventing the cytotoxic effect of palmitate are useful in reduction of diabetes complications. In this study, we introduced RSG as an agent that protects skeletal muscle cells against palmitate-induced apoptosis and insulin resistance. It appears that RSG protects skeletal muscle cells against palmitate-induced apoptosis via the PPARγ-dependent and PTP1B-independent mechanisms. Given the role of FFAs in skeletal muscle apoptosis, these findings support the idea that RSG can ameliorate diabetes complications such as skeletal muscle loss.

Protein tyrosine phosphatase 1B inhibition ameliorates palmitate-induced mitochondrial dysfunction and apoptosis in skeletal muscle cells.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin signaling pathway and is considered a promising therapeutic target in the treatment of diabetes. However, the role of PTP1B in palmitate-induced mitochondrial dysfunction and apoptosis in skeletal muscle cells has not been studied. Here we investigate the effects of PTP1B modulation on mitochondrial function and apoptosis and elucidate the underlying mechanisms in skeletal muscle cells. PTP1B inhibition significantly reduced palmitate-induced mitochondrial dysfunction and apoptosis in C2C12 cells, as these cells had increased expression levels of PGC-1α, Tfam, and NRF-1; enhanced ATP level and cellular viability; decreased TUNEL-positive cells; and decreased caspase-3 and -9 activity. Alternatively, overexpression of PTP1B resulted in mitochondrial dysfunction and apoptosis in these cells. PTP1B silencing improved mitochondrial dysfunction by an increase in the expression of SIRT1 and a reduction in the phosphorylation of p65 NF-κB. The protection from palmitate-induced apoptosis by PTP1B inhibition was also accompanied by a decrease in protein level of serine palmitoyl transferase, thus resulting in lower ceramide content in muscle cells. Exogenous addition of C2-ceramide to PTP1B-knockdown cells led to a reduced generation of reactive oxygen species (ROS), whereas PTP1B overexpression demonstrated an elevated ROS production in myotubes. In addition, PTP1B inhibition was accompanied by decreased JNK phosphorylation and increased insulin-stimulated Akt (Ser473) phosphorylation, whereas overexpression of PTP1B had the opposite effect. The overexpression of PTP1B also induced the nuclear localization of FOXO-1, but in contrast, suppression of PTP1B reduced palmitate-induced nuclear localization of FOXO-1. In summary, our results indicate that PTP1B modulation results in (1) alterations in mitochondrial function by changes in the activity of SIRT1/NF-κB/PGC-1α pathways and (2) changes in apoptosis that result from either a direct effect of PTP1B on the insulin signaling pathway or an indirect influence on ceramide content, ROS generation, JNK activation, and FOXO-1 nuclear translocation.

Protein tyrosine phosphatase 1B (PTP1B) modulates palmitate-induced cytokine production in macrophage cells.

OBJECTIVE: The aim of the study was to investigate the effect of PTP1B modulation on palmitate-induced cytokine production in macrophages. METHODS: Lentiviruses carrying PTP1B-shRNA or cDNA at different multiplicities of infection (MOIs) were used to decrease and increase PTP1B expression in Raw 264.7 cells, respectively. mRNA and protein levels of TNF-α and IL-6 were measured by real-time PCR and ELISA, respectively. RESULTS: 0.5 mM palmitate reduced PTP1B mRNA and protein levels by 25 and 19 %, respectively, compared to untreated cells. Overexpression of PTP1B decreased mRNA and protein levels of TNF-α and IL-6 in macrophages stimulated with palmitate. We found that protein and mRNA levels of cytokines significantly increased in knockdown cells stimulated by palmitate in a dose-dependent manner with increased MOI. NF-kB, JNK, p38 and ERK specific inhibitors significantly reduced the production of TNF-α and IL-6 in macrophages stimulated with palmitate and also PTP1B knockdown cells. Furthermore, inhibition of PTP1B resulted in increased phosphorylation of JNK, p38, ERK and NF-kB p65 in macrophage cells. CONCLUSIONS: The data of this study demonstrate that PTP1B negatively regulates palmitate-induced cytokine secretion in macrophages by mechanisms involving the activation of MAPKs and NF-kB pathways.

Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation.

Palmitate induces PTP1B expression in skeletal muscle cells. The purpose of this study was to investigate the mechanisms responsible for palmitate-induced PTP1B expression in mouse skeletal muscle cells. Three truncated fragments of PTP1B promoter were cloned into PGL3-basic vector and the promoter activity of PTP1B was assessed in C2C12 cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. EMSA was performed to examine binding of NF-κB to NF-κB consensus sequence and PTP1B oligonucelotides in the cells treated with palmitate. Lentiviral PTP1B-shRNA was used to knockdown PTP1B in myotubes. The phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. 0.5mM palmitate induced PTP1B promoter activity in fragment -1715/+59 by 50% (p<0.01). Palmitate increased NF-κB binding to both NF-κB consensus sequence and one NF-κB sequence (-920 to -935) in PTP1B promoter. Incubation of C2C12 cells with different concentrations of C2-ceramide enhanced PTP1B promoter activity dose-dependently. Inhibitors of de novo ceramide synthesis prevented palmitate-induced PTP1B promoter activity in myotubes. In addition, inhibitor of JNK pathway prevented ceramide-induced PTP1B promoter activity in myotubes. Knockdown of PTP1B also prevented ceramide-reduced IRS-1 and Akt phosphorylations in the myotubes. Exposure of the cells to PMA and calphostin C, an inhibitor of PKC, did not affect the activity of PTP1B promoter. Our data provide the evidence that the mechanism by which palmitate increased the expression of PTP1B seems to be through a mechanism involving the activation of ceramide-JNK and NF-κB pathways.

Palmitate and inflammatory state additively induce the expression of PTP1B in muscle cells.

The factors responsible for up-regulation of PTP1B, a negative regulator of insulin signaling, in insulin resistance state are not well understood. We performed a series of experiments in C2C12 muscle cells to determine the role of palmitate and an inflammatory state in regulation of PTP1B. Palmitate (0.75mM) induced PTP1B mRNA and protein level only at 16h. The combination of palmitate and macrophages, accompanied by a great increase of TNF-alpha and IL-6 in the culture media, additively caused a higher level of PTP1B protein levels in the muscle. Higher concentrations of palmitate reduced insulin stimulated glucose uptake in myotubes. A specific inhibitor of PTP1B partly increased insulin stimulated glucose uptake in palmitate treated cells. In conclusion, our results showing the additive influence of palmitate and the inflammatory state in the expression of PTP1B imply the involvement of these factors in the overexpression of PTP1B in insulin resistance state. We further provided the evidence suggesting the mediatory role for PTP1B in palmitate induced insulin resistance in myotubes.