An update on the secretory functions of brown, white, and beige adipose tissue: Towards therapeutic applications.

Adipose tissue, including white adipose tissue (WAT), brown adipose tissue (BAT), and beige adipose tissue, is vital in modulating whole-body energy metabolism. While WAT primarily stores energy, BAT dissipates energy as heat for thermoregulation. Beige adipose tissue is a hybrid form of adipose tissue that shares characteristics with WAT and BAT. Dysregulation of adipose tissue metabolism is linked to various disorders, including obesity, type 2 diabetes, cardiovascular diseases, cancer, and infertility. Both brown and beige adipocytes secrete multiple molecules, such as batokines, packaged in extracellular vesicles or as soluble signaling molecules that play autocrine, paracrine, and endocrine roles. A greater understanding of the adipocyte secretome is essential for identifying novel molecular targets in treating metabolic disorders. Additionally, microRNAs show crucial roles in regulating adipose tissue differentiation and function, highlighting their potential as biomarkers for metabolic disorders. The browning of WAT has emerged as a promising therapeutic approach in treating obesity and associated metabolic disorders. Many browning agents have been identified, and nanotechnology-based drug delivery systems have been developed to enhance their efficacy. This review scrutinizes the characteristics of and differences between white, brown, and beige adipose tissues, the molecular mechanisms involved in the development of the adipocytes, the significant roles of batokines, and regulatory microRNAs active in different adipose tissues. Finally, the potential of WAT browning in treating obesity and atherosclerosis, the relationship of BAT with cancer and fertility disorders, and the crosstalk between adipose tissue with circadian system and circadian disorders are also investigated.

Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells.

BACKGROUND: Extracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs. METHODS: Cell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins. RESULTS: We observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion. CONCLUSIONS: Our findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.

Small particles or vesicles released by cancer cells into their surroundings have the potential to stimulate the spread and growth of cancer cells. In this study, we focused on two specific molecules presented by these cancer cell-derived vesicles that could play a role in promoting the dissemination of cancer cells: a protein related to blood clotting and a protein on the cell surface.We found that large vesicles from bladder cancer cells that have the ability to spread had higher levels of these proteins than vesicles from nonspreading cancer cells. We also found that the former could make cancer cells move about more, produce more of a substance that helps cancer cells spread, and invade other tissues.To counteract the cancer-promoting actions of these vesicles, we examined the impact of combining a naturally occurring anticlotting protein that can be released by medications derived from heparin with an inhibitor targeting the cancer cell surface protein. We found that this combination stopped the vesicles from helping cancer cells move about more, produce more of the spreading substance, and invade other tissues.This approach of simultaneously targeting the two protein molecules present on cancer cell-derived vesicles might be a new way to treat bladder cancer.

Diagnostic Value of ATG5, Apo-Lipoprotein B-48, Thyroid Hormones, and Homocysteine in Patients with Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia. In this study, serum levels of autophagy-related 5 (ATG5), apolipoprotein B-48, thyroid hormones, and homocysteine were examined in patients with AD to determine their diagnostic and predictive value for early diagnosis and prevention of AD. MATERIALS: For this study, fifty serum samples were obtained from patients with AD and fifty serum samples from healthy controls. Serum levels of ATG 5, apo B48, thyroid hormones, homocysteine, vitamin B12, and folic acid were determined by ELISA. Spectrophotometry was used to determine serum lipid concentrations. RESULTS: The mean age of the case group was 69 ± 6.4 years and that of the control group was 67 ± 4.2 years. There were differences between the control and case groups in serum levels of homocysteine, apo B48, ATG5, hsCRP, LDL, HDL, cholesterol, and VitB12 (p < 0.05). According to the results of the ROC curve, measurements of serum levels of ATG5, homocysteine, and apo B48 have excellent performance in distinguishing patients with Alzheimer's disease from patients without AD. CONCLUSIONS: This study suggests that the measurement of serum levels of ATG5, homocysteine, and apo B48, along with other available biomarkers, can be helpful in the diagnosis and management of patients with AD in the early stages of their disease.

Fatty acids of type 2 diabetic serum decrease the stemness properties of human adipose-derived mesenchymal stem cells.

In type 2 diabetes, dyslipidemia and increased serum free fatty acids (FFAs) exacerbate the development of the disease through a negative effect on insulin secretion. Adipose-derived mesenchymal stem cells (AdMSCs) play a key role in regenerative medicine, and these cells can potentially be applied as novel therapeutic resources in the treatment of diabetes. In this study, AdMSCs were treated with diabetic or nondiabetic serum FFAs isolated from women of menopausal age. Serum FFAs were analyzed using gas-liquid chromatography. The expression level of the stemness markers CD49e and CD90 and the Wnt signaling target genes Axin-2 and c-Myc were evaluated using real-time PCR. The proliferation rate and colony formation were also assessed using a BrdU assay and crystal violet staining, respectively. The level of glutathione was assessed using cell fluorescence staining. Compared to nondiabetic serum, diabetic serum contained a higher percentage of oleate (1.5-fold, p < 0.01). In comparison with nondiabetic FFAs, diabetic FFAs demonstrated decreasing effects on the expression of CD90 (-51%, p < 0.001) and c-Myc (-48%, p < 0.05), and proliferation rate (-35%, p < 0.001), colony formation capacity (-50%, p < 0.01), and GSH levels (-62%, p < 0.05). The negative effect of the FFAs of diabetic serum on the stemness characteristics may impair the regenerative capabilities of AdMSCs.

The porcupine inhibitor WNT974 provokes ectodermal lineage differentiation of human embryonic stem cells.

Porcupine (Porcn) enzyme plays an essential role in Wnt signaling activation. Stearoyl-CoA desaturase-1 (SCD1) is required to provide Porcn substrates. The aim of this study was to determine the effect of a novel Porcn inhibitor on the fate of human embryonic stem cells (hESCs) and the reliance of Porcn on SCD1 activity. hESCs were cultured on a feeder layer or Matrigel-coated plates. Small molecules WNT974 (LGK-974) and CAY10566 were used to inhibit Porcn and SCD1 activity, respectively. We assessed the effect of Porcn inhibition on viability, expression of Wnt signaling targets, pluripotency markers, proliferation, differentiation, and protein fatty acylation. hESCs' conditioned medium (CM) containing secreted Wnt proteins were applied in rescue experiments. To examine the catalytic dependency of Porcn on SCD1, the results of combined inhibitor treatment were compared with the SCD1 inhibitor alone. LGK-974 at the selected concentrations showed mild effects on hESCs viability, but significantly reduced messenger RNA and protein expression of Wnt signaling targets (Axin-2 and c-Myc) and pluripotency markers (OCT-4 and SOX-2) (p < .05). Adding 1 µM of Porcn inhibitor reduced proliferation (p = .03) and enhanced differentiation capacity into ectodermal progenitors (p = .02), which were reverted by CM. Click chemistry reaction did not show significant alteration in protein fatty acylation upon LGK-974 treatment. Moreover, combined inhibitor treatment caused no further substantial reduction in Wnt signaling targets, pluripotency markers, and protein fatty acylation relative to CAY10566-treated cultures. The substrate availability for Porcn activity is regulated by SCD1 and targeting Porcn by LGK-974 prompts the transition of hESCs from self-renewal state to ectodermal lineage.

Steroid-depleted endometriosis serum improves oocyte maturation in IVM systems.

In vitro maturation (IVM) is a novel approach to overcome the adverse effects of human in vitro fertilization (IVF). The aim of the present study is to evaluate the effect of total and steroid-depleted serum obtained from patients with endometriosis on IVM outcome as supplementation for this system. To this purpose, patients with endometriosis were selected according to in/excluding criteria. Germinal vesicles (GVs) and cumulus cells were treated with 10% of each serum. The expression levels of stearoyl CoA desaturase 1 (SCD 1) and cyclooxygenase-2 (COX-2) genes were evaluated by RT-qPCR. Gas-liquid chromatography and flow cytometry were performed to analyze fatty acids composition and apoptosis. The mRNA expression levels of SCD1 (2.47 fold) and COX-2 (6.4 fold), and also the synthesis of oleate, linoleate, and arachidonate were increased (1.19, 1.06, and 2.37 folds, respectively) in cumulus cells treated with steroid-depleted serum (p < .05). The synthesis of palmitate, palmitoleate, and stearate (0.995, 0.67, and 0.7 folds, respectively) and also the rate of apoptosis were significantly decreased in these cells (p < .05). Moreover, GVs cultured in steroid-depleted group showed a significantly higher rate of maturation (p < .001). Overall, our findings imply a new insight into the expansion of IVM system in oocytes development.

Activation of toll-like receptor signaling in endothelial progenitor cells dictates angiogenic potential: from hypothesis to actual state.

Human endothelial progenitor cells (EPCs) were isolated from cord blood samples and enriched by magnetic activated cell sorting method based on the CD133 marker. Cells were incubated with different doses of bacterial lipopolysaccharide, ranging from 2, 5, 10, 50, 100, 200, 250, 500, to 1000 µg/ml, for 48 h. The cell survival rate was determined by using MTT assay. To confirm activation of the toll-like receptor signaling pathway, PCR array analysis was performed. Protein levels of ERK1/2, p-ERK1/2, NF-ƙB and TRIF proteins were measured using western blotting. The content of TNF-α and lipoprotein lipase activity were analyzed by immunofluorescence imaging. Flow cytometric analysis of CD31 was performed to assess the maturation rate. Cell migration was studied by the Transwell migration assay. The expression of genes related to exosome biogenesis was measured using real-time PCR analysis. In vivo gel plug angiogenesis assay was done in nude mice. Lipopolysaccharide changed endothelial progenitor cells' survival in a dose-dependent manner with maximum viable cells in groups treated with 2 µg/ml. PCR array analysis showed the activation of toll-like signaling pathways after exposure to LPS (p<0.05). Western blotting analysis indicated an induction of p-ERK1/2 and Erk1/2, NF-kB and TRIF in LPS-treated EPCs compared with the control (p<0.05). Immunofluorescence staining showed an elevation of TNF-α and lipoprotein lipase activity after lipopolysaccharide treatment (p<0.05). Lipopolysaccharide increased EPC migration and expression of exosome biogenesis-related genes (p<0.05). In vivo gel plug analysis revealed enhanced angiogenesis in cells exposed to bacterial lipopolysaccharide. Data highlighted the close relationship between the toll-like receptor signaling pathway and functional activity in EPCs.

Fatty acids of follicular fluid phospholipids and triglycerides display distinct association with IVF outcomes.

RESEARCH QUESTION: Are triglyceride fatty acids in the follicular fluid associated with either follicular fluid phospholipid fatty acids or IVF outcomes and, if so, how are they associated? DESIGN: In a prospective cross-sectional study, 70 women undergoing intracytoplasmic sperm injection were recruited. Follicular fluid phospholipids and triglycerides were separated by thin-layer chromatography. Fatty acids were measured using gas-liquid chromatography and flame ionization detection system. RESULTS: Significant differences in fatty acid composition were observed between follicular fluid phospholipid and triglyceride fractions. Phospholipid stearic acid and n-3 polyunsaturated fatty acids, particularly alpha-linolenic acid, were negatively associated with the number of mature oocytes and cleaved embryos, whereas arachidonic acid was in direct correlation with cleavage rate per IVF cycle (ß = 0.325, P = 0.022). In the case of triglyceride fraction, total monounsaturated fatty acids, oleic acid in particular, displayed significantly positive associations with the number of oocytes (ß = 0.261, P = 0.043) and embryos (ß = 0.310, P = 0.018). Furthermore, cleavage rate correlated inversely with palmitic acid (ß = -0.359, P = 0.007) and directly with pentadecanoic acid (ß = 0.378, P = 0.005). Most of these associations, however, were not independent of predictive fatty acids belonging to phospholipid fraction, according to multivariate analysis. CONCLUSIONS: Fatty acid compositions of phospholipid and triglyceride fractions from human follicular fluid differentially correlate with IVF cycle parameters.

Arachidonic acid alleviates the detrimental effects of acetylsalicylic acid on human granulosa cells performance in vitro.

Here, we investigated the biological effects of arachidonic acid (AA) in human cumulus granulosa cells (CGCs) after exposure to ASA. Cells were isolated from the follicular fluid and incubated with 0.5 mM acetylsalicylic acid (ASA) and 50 µM AA. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. E2 and P4 levels were measured by chemiluminescence assay. Expression of genes including CYP19A1, FACN, and SCD1 was measured by real-time polymerase chain reaction assay. Oxidative status was analyzed by monitoring glutathione peroxidase activity. The fatty acid profile was analyzed by the gas chromatography technique. Enzyme-linked immunosorbent assay was used to measure prostaglandin E2 (PGE2 ) in CGCs after exposure to ASA and AA. Protein levels of the estrogen receptor were studied by immunofluorescence staining. Ultrastructural changes were evaluated by transmission electron microscopy imaging. ASA treatment reduced E2 production, Cyp19a1 expression, glutathione peroxidase (GPx) activity, and estradiol receptor expression in CGCs. The addition of AA prevented the ASA-induced E2 reduction (p < .05) and expression of Cyp19a1. Moreover, AA increased the antioxidant capacity of CGCs exposed to ASA by promoting GPx activity (p < .05). AA increased monounsaturated fatty acid/saturated fatty acid ratio compared with the ASA group (p < .05). AA supplementation triggered the synthesis and secretion of PGE2 in ASA-treated CGCS (p < .05). Cytoplasmic vacuolation observed in the ASA group and treatment with AA intensified vacuolation rate. The expression of the estrogen receptor was increased after AA supplementation. Data demonstrated that AA decreased the detrimental effects of ASA on human CGCs after 72 hr.

Fractalkine and apoptotic/anti-apoptotic markers in granulosa cells of women with polycystic ovarian syndrome.

Owing to the role of fractalkine in regulating cellular apoptosis/proliferation, we investigated fractalkine effects on apoptosis/proliferation signaling of granulosa cells in polycystic ovarian syndrome (PCOS) patients through in vitro and in vivo experiments. In vivo, granulosa cells were collected from 40 women undergoing oocyte retrieval (20 controls and 20 PCOS). The expression levels of fractalkine, BAX, Bcl2, Bcl2-XL, Bad, and TNF-α were assessed using RT-PCR. In vitro, we determined the effect of different doses of fractalkine on the expression of the above mentioned genes in GCs of both groups. We found that the expression levels of fractalkine and Bcl-2 were significantly lower in the GCs of PCOS patients compared to the control group (p < 0.05). In contrast, the expression levels of TNF-α and BAX were higher in the patient's group than in the control group. The results suggested that expression levels of fractalkine were negatively and positively correlated with the number of oocytes and fertilized oocytes respectively. Moreover, fractalkine could dose-dependently increase fractalkine and decrease BAD, BAX, Bcl-xl, and TNF-α expressions in the control GCs. In contrast, GCs collected from PCOS patients revealed an increase in expression of BAD, BAX, and Bcl-xl following fractalkine treatment. Our findings indicated that insufficient expression of fractalkine in PCOS patients is related with elevated apoptotic and inflammatory markers and reduced anti-apoptotic genes in the GCs.