The size of human subcutaneous adipocytes, but not adiposity, is associated with inflammation, endoplasmic reticulum stress, and insulin resistance markers.

BACKGROUND: The fat storage capacity of the adipose tissue prevents ectopic lipid deposition, which is one of the risk factors for metabolic abnormalities in obesity. This capacity depends upon the adipogenic gene expression and blood supply provision for tissue expansion through angiogenesis. Here, we studied hyperplasia/hypertrophy of subcutaneous white adipose tissue (scWAT) concerning adipogenic gene expression, angiogenic status, and metabolic parameters in non-obese and different classes of obese individuals. METHODS: The scWAT samples were collected from 80 individuals. The anthropometric parameters, adipose tissue cell size, serum biochemistry, ER stress-induced XBP1 splicing, PPARγ2, SFRP1, WNT10B, and VEGFA gene expression levels were studied. In addition, the CD31 level was investigated by Western blotting. RESULTS: The obese individuals had greater waist circumferences and higher serum TG, TC, insulin, and HOMA-IR than the non-obese group. However, the largest adipocyte size, increased TNFα, insulin, and HOMA-IR, and the highest expression level of sXBP1, WNT10B, and VEGFA were observed in Class I obese individuals. It means that inflammation, insulin resistance, and ER stress accompany hypertrophic scWAT adipocytes with limited adipose tissue expansion ability. Furthermore, the Class II + III obese individuals showed high PPARγ2 expression and CD31 levels. There is adipogenesis through hyperplasia in this group. The SFRP1 expression was not significantly different in the studied groups. CONCLUSION: The results suggest that the capability of adipogenesis with inadequate angiogenesis is related to the metabolic status, inflammation, and ER function. Therefore, therapeutic strategies that support both angiogenesis and adipogenesis can effectively prevent the complications of obesity.

Protective effect of saffron carotenoids against amyloid beta-induced neurotoxicity in differentiated PC12 cells via the unfolded protein response and autophagy.

The preventive effect of saffron against Alzheimer's disease (AD) has been reported. Herein, we studied the effect of Cro and Crt, saffron carotenoids, on the cellular model of AD. The MTT assay, flow cytometry, and elevated p-JNK, p-Bcl-2, and c-PARP indicated the AßOs-induced apoptosis in differentiated PC12 cells. Then, the protective effects of Cro/Crt on dPC12 cells against AßOs were investigated in preventive and therapeutic modalities. Starvation was used as a positive control. RT-PCR and Western blot results revealed the reduced eIF2α phosphorylation and increased spliced-XBP1, Beclin1, LC3II, and p62, which indicate the AßOs-induced autophagic flux defect, autophagosome accumulation, and apoptosis. Cro and Crt inhibited the JNK-Bcl-2-Beclin1 pathway. They altered Beclin1 and LC3II and decreased p62 expressions, leading cells to survival. Cro and Crt altered the autophagic flux by different mechanisms. So, Cro increased the rate of autophagosome degradation more than Crt, while Crt increased the rate of autophagosome formation more than Cro. The application of 4µ8C and chloroquine as the inhibitors of XBP1 and autophagy, respectively, confirmed these results. So, augmentation of the survival branches of UPR and autophagy is involved and may serve as an effective strategy to prevent the progression of AßOs toxicity.

Cadmium nanocluster as a safe nanocarrier: biodistribution in BALB/c mice and application to carry crocin to breast cancer cell lines.

Aim: Metal nanoclusters are emerging nanomaterials applicable for drug delivery. Here, the toxicity and oxidative stress induction of divalent cationic cadmium (Cd2+) was compared with a Cd in the form of nanocluster. Then, it was used for targeted drug delivery into breast cancer cell lines. Methods: Using a green chemistry route, a Cd nanocluster (Cd-NC) was synthesized based on bovine serum albumin. After characterization, its genotoxicity and oxidative stress induction were studied in both in vitro and in vivo. After that, it was conjugated with hyaluronic acid (HA). The efficiency of hyaloronized-Cd-CN (HA-Cd-NC) for loading and releasing crocin (Cro), an anticancer phytochemical, was studied. Finally, it was applied for cell death induction in a panel of breast cancer cell lines. Results: The comet assay results indicated that, unlike Cd2+ and potassium permanganate (KMnO4), no genotoxicity and oxidative stress was induced by Cd-NC in vitro. Then, the pharmacokinetics of this Cd-NC was studied in vivo. The data showed that Cd-NC has accumulated in the liver and excreted from the feces of mice. Unlike Cd2+, no toxicity and oxidative stress were induced by this Cd-NC in animal tissues. Then, the Cd-NC was targeted toward breast cancer cells by adding HA, a ligand for the CD44 cell surface receptor. After that, Cro was loaded on HA-Cd-NC and it was used for the treatment of a panel of human breast cancer cell lines with varying degrees of CD44. The half-maximal drug inhibitory concentration (IC50) of Cro was significantly decreased when it was loaded on HA-Cd-NC, especially in MDA-MB-468 with a higher degree of CD44 at the surface. These results indicate the higher toxicity of Cro toward breast cancers when carried out by HA-Cd-NC. Conclusions: The Cd-NC was completely safe and is a promising candidate for delivering anticancer drugs/phytochemicals into the targeted breast tumors.

The Effect of Ultrasonication on the Fibrillar/ Oligomeric Structures of Aß1-42 at Different Concentrations.

The number of disease states linked the aberrant regular protein conformations to oligomers and amyloid fibrils. Amyloid beta 1-42 (Aß1-42) peptide is very hydrophobic and quickly forms the ß-rich structure and fibrillar protein aggregates in some solutions and buffer conditions. Ultrasonication pulses can disrupt amyloid fibrils to smaller fragments and produce Aß1-42 peptides of different sizes and oligomers. Herein, we investigated the effects of buffer and ultrasonication on Aß1-42 structure at low and high concentrations. After ultrasonication, the Western blot results showed that Aß1-42 fibrils were disaggregated into different sizes. The transmission electron microscopy results indicated Aß1-42 at low concentration (25 µM) in Ham's/F12 phenol red-free culture medium formed short-size fragments and oligomers. In comparison, Aß1-42 at higher concentration (100 µM) formed fibrils that break down into smaller fragments after ultrasonication. However, after regrowth, it formed mature fibrils again. Cell viability assay indicated that Aß1-42 oligomers formed at a low concentration (25 µM) were more toxic to PC12 cells than other forms. In conclusion, by applying ultrasonication pulses and controlling peptide concentration and buffer condition, we can rich Aß1-42 aggregates with a particular size and molecular structure.

Saffron carotenoids reversed the UCMS-induced depression and anxiety in rats: Behavioral and biochemical parameters, and hippocampal BDNF/ERK/CREB and NR2B signaling markers.

BACKGROUND: Depression is a debilitating condition that affects the mind and the individual's body. The improving effects of saffron on depression and anxiety have long been discussed, with limited information about the molecular mechanism of action. HYPOTHESIS/PURPOSE: Investigating the effect of saffron carotenoids, Crocin and Crocetin, on depression and anxiety in rats by emphasizing some signaling pathways involved. STUDY DESIGN: Depression and anxiety were induced in rats via unpredictable chronic mild stress (UCMS). Then different rat groups were treated with Crocin, Crocetin, Fluoxetine, and vehicle. Behavioral tests were done before and after treatment. METHODS: The serum Serotonin and Corticosterone and the expression of some hippocampal signaling proteins were studied. Furthermore, bioinformatics tools were used to predict the interactions of Crocin/ Crocetin with the Serotonin transporter and NMDA receptor subunit NR2B. Then, the patch-clamp was used to study the interaction of Crocetin with the NMDA receptor. RESULTS: Various behavioral tests confirmed the induction of depression and the improvement of depression by these natural carotenoids. In addition, Crocin/ Crocetin significantly increased the decreased serum Serotonin and reduced the increased serum Corticosterone in the depressed groups. They also increased or caused a trend of increase in the CREB, ERK, BAD, BDNF, p11, and 5-HT1B expression in the hippocampus of the depressed groups. In addition, there were an increase or a trend in p-CREB/CREB, p-ERK1/2 /ERK1/2, and p-BAD/BAD ratios in the Crocin/ Crocetin treated depressed groups. However, the NR2B and FOXO3a expression showed a trend of decrease in depressed groups after treatment. The bioinformatics data indicated that Crocin/ Crocetin could bind to the Serotonin transporter (SLC6A4) and NR2B subunit of the NMDA receptor. Both carotenoids bind to the same site as Fluoxetine in the SLC6A4. However, they bound to different sites on the NR2B. So, Crocetin binds to NR2B at the same site as Ifenprodil. But Crocin bound to another site. The whole cell patch-clamp recording on the normal rat hippocampus revealed a significant decrease in the NMDA peak amplitude after Crocetin treatment, indicating its inhibitory effect on this receptor. CONCLUSION: The antidepressant activities of Crocin/ Crocetin are possibly due to their effects on Serotonin and Corticosterone serum concentrations, NR2B expression, and the downstream signaling pathways. Furthermore, these natural carotenoids, like Fluoxetine, induced an increasing tendency in p11 and 5HT1B in depressed rats.

Possible role of WNT10B in increased proliferation and tubule formation of human umbilical vein endothelial cell cultures treated with hypoxic conditioned medium from human adipocytes.

Regulation of angiogenesis plays an important role in adipose tissue expansion and function. The Wnt pathway and WNT10B, the main member of Wnt family, participate in angiogenesis in cancer tumors, but there is limited evidence to support the regulatory role of WNT10B in human adipose tissue angiogenesis. Subcutaneous white adipose tissue (scWAT) of 80 participants including obese and non-obese subjects was obtained and the expression of WNT10B and VEGFA genes were evaluated using qPCR. Human adipose-derived stem cells (hADSC) were differentiated to adipocytes and incubated under either hypoxic or normoxic conditions. The conditioned media of these adipocytes were collected and used as growth media for human umbilical vein endothelial cells (HUVEC) in Matrigel. We evaluated the proliferation, cell cycle phases, tubule formation and ß-catenin activation of these treated cells. We found a significant correlation between WNT10B and VEGFA expression in the scWAT of both obese and non-obese subjects. Proliferation and tubule formation of HUVEC treated with conditioned media of hypoxic adipocytes (hCM) in the S-phase were increased significantly compared to the HUVEC treated with the conditioned media of normoxic adipocytes (nCM). The expression of WNT10B and VEGFA was enhanced in hypoxic adipocytes compared to normoxic adipocytes; also, activation and nuclear translocation of ß-catenin was enhanced in the HUVEC treated with hCM compared to nCM. WNT10B acts as an angiogenic protein in scWAT under hypoxic conditions. Hypoxia induced WNT10B increases VEGFA expression and causes tube formation by HUVECs and angiogenesis in adipose tissue via the canonical Wnt/ß-catenin pathway.

Proteomics analysis revealed the presence of inflammatory and oxidative stress markers in the plasma of migraine patients during the pain period.

BACKGROUND: There is increasing evidence that some biomarkers are implicated in migraine pathogenesis. This study looks at plasma proteome in migraine patients for potential protein biomarkers. METHODS: This case-control study has two phases. In phase I, plasma samples were collected from three groups, including twenty-three episodic migraineurs, thirty-five chronic migraineurs, and twenty-nine healthy subjects. In phase II, plasma samples were prepared from two groups, including five episodic and five chronic migraine cases, during the pain and 24 h after the pain-free periods. Two-dimensional gel electrophoresis (2-DE) was performed on plasma proteins. The possible corresponding proteins for the differentially expressed spots between groups investigated by the Melanie software were predicted by 2-DE gels of the EXPASY database. LC-MS/MS additionally analyzed phase II data. RESULTS: Expression levels of haptoglobin, clusterin, fibrinogen alpha chain, fibrinogen beta chain, complement c3, transthyretin, α1-microglobulin, and retinol-binding protein 4 were shown considerable changes in migraine patients compared to controls or their pain-free period. CONCLUSION: Differences in expression levels for several proteins were observed across groups. Most of these are associated with inflammation, oxidative stress, and neuroprotection, which can be considered potential disease biomarkers. However, further research is necessary for this respect.

L-lysine protects C2C12 myotubes and 3T3-L1 adipocytes against high glucose damages and stresses.

Hyperglycemia is a hallmark of diabetes, which is associated with protein glycation and misfolding, impaired cell metabolism and altered signaling pathways result in endoplasmic reticulum stress (ERS). We previously showed that L-lysine (Lys) inhibits the nonenzymatic glycation of proteins, and protects diabetic rats and type 2 diabetic patients against diabetic complications. Here, we studied some molecular aspects of the Lys protective role in high glucose (HG)-induced toxicity in C2C12 myotubes and 3T3-L1 adipocytes. C2C12 and 3T3-L1 cell lines were differentiated into myotubes and adipocytes, respectively. Then, they were incubated with normal or high glucose (HG) concentrations in the absence/presence of Lys (1 mM). To investigate the role of HG and/or Lys on cell apoptosis, oxidative status, unfolded protein response (UPR) and autophagy, we used the MTT assay and flow cytometry, spectrophotometry and fluorometry, RT-PCR and Western blotting, respectively. In both cell lines, HG significantly reduced cell viability and induced apoptosis, accompanying with the significant increase in reactive oxygen species (ROS) and nitric oxide (NO). Furthermore, the spliced form of X-box binding protein 1 (XBP1), at both mRNA and protein levels, the phosphorylated eukaryotic translation initiation factor 2α (p-eIf2α), and the Light chain 3 (LC3)II/LC3I ratio was also significantly increased. Lys alone had no significant effects on most of these parameters; but, treatment with HG plus Lys returned them all to, or close to, the normal values. The results indicated the protective role of Lys against glucotoxicity induced by HG in C2C12 myotubes and 3T3-L1 adipocytes.

Isolation and characterization of the primary epithelial breast cancer cells and the adjacent normal epithelial cells from Iranian women's breast cancer tumors.

As an experimental model, most studies rely on established human cancer cell lines; however, some genetical or phenotypical differences exist between these cells and their original tumor. Therefore, primary cells isolated directly from tissue are believed to be more biologically relevant tools for studying human and animal biology. Here, we aimed to isolate primary epithelial cancer and normal cells from breast tumors of Iranian women, for the first time. Thus, we isolated the epithelial and fibroblast cells from biopsy samples of patients with breast cancer based on differential centrifugation followed by culture in selective media. Normal epithelial cells obtained from the tissue biopsy away from the core of the tumor, based on the pathological diagnosis. Flow cytometry analysis indicated the positive immunoreactivity of the isolated epithelial cells against CD24 and Epithelial Specific Antigen (ESA/EpCAM), while they displayed a concomitant low expression of CD44 and CD49f. In contrat to fibroblasts, the qPCR data indicated the expression of luminal intracellular cytokeratin (Ck18) in both normal and cancer epithelial cells, but there was no expression of myoepithelial/basal markers, CK5 and vimentin. The epithelial cancer cells were reactive to cytokeratin 19 (CK19) antibody, whereas the normal epithelial cells were not. The expression of calmodulin-like protein (CLP) was also lower in the cancer epithelial cells than in the normal ones. In conclusion, primary epithelial normal and cancer cells, in addition to the fibroblasts were isolated and characterized from breast tumor of Iranian patients; and CLP expression is suggested as a susceptibility marker for breast cancer screening.