The up-regulation of markers of adipose tissue fibrosis by visfatin in pre-adipocytes as well as obese children and adolescents.

Adipocytes are surrounded by a three-dimensional network of extracellular matrix (ECM) proteins. Aberrant ECM accumulation and remodeling leads to adipose tissue fibrosis. Visfatin is one of the adipocytokines that is increased in obesity and is implicated in insulin resistance. The objective of this study was to investigate the effect of visfatin on major components of ECM remodeling. In this study, plasma levels of both endotrophin and visfatin in obese children and adolescents were significantly higher than those in control subjects and they showed a positive correlation with each other. Treatment of 3T3-L1 pre-adipocytes with visfatin caused significant up-regulation of Osteopontin (Opn), Collagen type VI (Col6), matrix metalloproteinases MMP-2 and MMP-9. By using inhibitors of major signaling pathways it was shown that visfatin exerted its effect on Col6a3 gene expression through PI3K, JNK, and NF-кB pathways, while induced Opn gene expression via PI3K, JNK, MAPK/ERK, and NOTCH1. Our conclusion is that, the relationship between visfatin, endotrophin and insulin resistance parameters in obesity as well as increased expression of ECM proteins by visfatin suggests adipose tissue fibrosis as a mechanism for devastating effects of visfatin in obesity.

Descending Expression of miR320 in Insulin-Resistant Adipocytes Treated with Ascending Concentrations of Metformin.

Some miRNAs are supposed to play a role in insulin resistance and metabolic disorders. Such miRNAs can be differentially expressed in response to a pharmacologic intervention for insulin resistance as a biomarker/risk factor for insulin resistance. This study aimed at determining the effect of Metformin on miR320 expression in insulin-resistant (IR) adipocytes. The 3T3L1 cells were expanded in DMEM, differentiated into adipocytes by differentiating medium, became resistant to insulin, and then were treated with ascending concentrations of Metformin. Quantitative real-time PCR was performed to profile the miR320 expression in 3T3L1 adipocytes, IR adipocytes, and Metformin-treated IR adipocytes. Compared to the normal adipocytes, IR adipocytes exhibited a significantly higher level of miR320 expression, however, in response to Metformin graded concentrations, IR adipocytes down-regulated miR320 and were almost at normal level. The maximum effect of Metformin was at 10 mM. In IR adipocytes, miR320 expression is over-expressed which can be down-regulated by Metformin treatment. The findings provide some information on a potentially new marker to determine insulin resistance and to predict response to insulin resistance therapy.

Metformin reduces fibrosis factors in insulin resistant and hypertrophied adipocyte via integrin/ERK, collagen VI, apoptosis, and necrosis reduction.

AIMS: Fibrosis as the hallmark of adipose tissue dysfunction which is associated with insulin resistance and type 2 diabetes, results from deposition of excess extra cellular matrix components like collagen and increased cell death. Here we investigated the effect of antidiabetic drug, Metformin, on the factors that play role in fibrosis such as; integrin/ERK pathway, collagen VI, MMP2, MMP9, apoptosis markers including DAPK1, DAPK3, DAP, SIVA, necrosis markers including RIPK1, RIPK3, and MLKL in insulin resistant and hypertrophied adipocytes. METHODS: 3T3-L1 adipocytes after differentiation to insulin resistant and hypertrophied cells, treated with Metformin, and the gene expression of aforementioned factors assayed by real time PCR. The protein expression of collagen VI and ERK assayed by western blotting. KEY FINDINGS: The expression of integrins changed from 0.5 to 6-fold in hypertrophied adipocyte versus adipocyte. Apoptosis and necrosis markers increased >1.5-fold in insulin resistant and hypertrophied adipocytes. Also ECM components and ERK activation increased >2-fold and 1.5-fold, respectively in insulin resistant and hypertrophied adipocytes. Metformin caused reduction of activity of integrin/ERK pathway in Metformin treated insulin resistant and hypertrophied adipocytes compared to untreated group. Metformin also reduced collagen VI in both gene and protein expression level, MMP2 and MMP9 in gene expression, and also the expression of apoptosis and necrosis gene. SIGNIFICANCE: Metformin with reduction of ECM component as collagen VI, MMP2 and MMP9, integrin/ERK pathway, necrosis markers as RIPK1, RIPK3 and MLKL, and apoptosis markers including DAP, DAPK1, DAPK3 and SIVA effects on fibrosis in insulin resistant and hypertrophied adipocytes in vitro.

Gene expression profile evaluation of integrins in 3T3-L1 cells differentiated to adipocyte, insulin resistant and hypertrophied cells.

Integrins are cell attachment receptors that function in the communication between the intracellular and extracellular compartments. Integrins and extra cellular matrix (ECM) collaborate to regulate gene expression by extracellular signal-regulated kinases (ERKs). Integrins as regulators, have critical role in ECM remodeling. Fibrosis is the hallmark of obesity and insulin resistance originated by aberrant ECM remodeling. Therefore deciphering integrins' expression profile in cells at different conditions is worthy. The aim of this study is evaluation of integrins' gene expression profile changes in mouse 3T3-L1 preadipocytes, adipocytes, insulin resistant and hypertrophic adipocytes. For this purpose, we differentiated mouse 3T3-L1 preadipocytes to adipocytes, insulin resistant and hypertrophied adipocytes and assayed integrins' gene expression in four conditions by real time-PCR. Also the proteins expression changes of ERK and collagen VI assayed by Western blotting. Data analysis has shown that integrins' gene expression changes throughout adipocyte differentiation and pathological processes. The expressions of many integrins genes were significantly up- or down-regulated by >1.5-fold during differentiation, insulin resistant, and hypertrophic adipocytes. In addition to changes in the type of integrin, the integrins expression levels were different. Integrins, on the whole were more expressed in pathological processes relative to normal adipocytes. Also, phosphorylation of ERK 1,2 was increased >1.5-fold in differentiated, insulin resistant and hypertrophied adipocytes versus preadipocytes. Collagen VI only increased 2-fold in hypertrophied adipocytes. Examination of the total integrin gene family expression during adipocyte differentiation and pathological processes, leads to the identification of differential integrin gene expression. These results suggest that the type of integrin may not only play a role in adipocyte differentiation but also in pathological processes which may associate to increased ERK pathway activity in these conditions.

Development and characterization of a novel cationic PEGylated niosome-encapsulated forms of doxorubicin, quercetin and siRNA for the treatment of cancer by using combination therapy.

The aim of this study was to optimize the cationic PEGylated niosome-containing anti-cancer drugs and siRNA to enhance the therapeutic response. Therefore, various surfactant-based (tween-60) vesicles of doxorubicin (DOX; a chemotherapeutic drug) and quercetin (QC; a chemosensitizer) were prepared. To load siRNA on niosomes, 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) was used as a cationic lipid. The optimum formulation containing tween-60:cholesterol:DPPC:DOTAP:DSPE-PEG2000 at 49.5:5.5:15:25:5 demonstrated that the vesicle size and zeta potential were 52.8 ± 2.7 nm and +27.4 ± 2.3 mV, respectively. Entrapment efficiency (EE%) for DOX and QC was 86.4 ± 2.1% and 94.9 ± 3.9%, respectively. Moreover, the drug release during 6 h was 32.1 ± 1.6% and 30.5 ± 1.3% for DOX and QC, respectively denoted on the controlled release. The gel retardation assay demonstrated that siRNA could be successfully loaded into a cationic niosome:siRNA in a weight ratio 40:1. Additionally, noisome-encapsulated drugs had a higher toxicity against cancer cells when compared with un-encapsulated forms and the synergistic effects of co-delivery of siRNA and DOX with QC on gastric, prostate, breast cancer cells as well as human foreskin fibroblast as a normal cell line was shown. The results showed that the co-delivery of drugs and siRNA using cationic PEGylated niosomes exhibited an increased anti-cancer activity against the tumor cell death. It seems that cationic PEGylated niosomes have opened up a new avenue to enrich the armamentarium of therapeutic agents to fight cancer.

Selenium and its relationship with selenoprotein P and glutathione peroxidase in children and adolescents with Hashimoto's thyroiditis and hypothyroidism.

The essential trace element selenium (Se) is required for thyroid hormone synthesis and metabolism. Selenoproteins contain selenocysteine and are responsible for biological functions of selenium. Glutathione peroxidase (GPx) is one of the major selenoproteins which protects the thyroid cells from oxidative damage. Selenoprotein P (SePP) is considered as the plasma selenium transporter to tissues. The aim of this study was to evaluate serum Se and SePP levels, and GPx activity in erythrocytes of children and adolescents with treated Hashimoto's thyroiditis, hypothyroidism, and normal subjects. Blood samples were collected from 32 patients with Hashimoto's thyroiditis, 20 with hypothyroidism, and 25 matched normal subjects. All the patients were under treatment with levothyroxine and at the time of analysis all of the thyroid function tests were normal. GPx enzyme activity was measured by spectrophotometry at 340 nm. Serum selenium levels were measured by high-resolution continuum source graphite furnace atomic absorption. SePP, TPOAb (anti-thyroid peroxidase antibody), and TgAb (anti-thyroglobulin antibody) were determined by ELISA kits. T4, T3, T3 uptake and TSH were also measured. Neither GPx activity nor SePP levels were significantly different in patients with Hashimoto's thyroiditis or hypothyroidism compared to normal subjects. Although GPx and SePP were both lower in patients with hypothyroidism compared to those with Hashimoto's thyroiditis and normal subjects but the difference was not significant. Serum Se levels also did not differ significantly in patients and normal subjects. We did not find any correlation between GPx or SePP with TPOAb or TgAb but SePP was significantly correlated with Se. Results show that in patients with Hashimoto's thyroiditis or hypothyroidism who have been under treatment with levothyroxine and have normal thyroid function tests, the GPx, SePP and Se levels are not significantly different.

S-Allylcysteine, a garlic compound, increases ABCA1 expression in human THP-1 macrophages.

ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cholesterol efflux to apoA-I in lipid-loaded macrophages, which is the first step of reverse cholesterol transport in vivo and a critical step in preventing atherosclerosis. Enhanced ABCA1 expression may inhibit foam cell formation and consequently reduce atherogenic risk. The purpose of this study was to investigate the effect of S-allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract, on the expression of ATP-binding cassette transporter A1 in human THP-1 macrophages. The human monocyte THP-1 cells were differentiated to macrophage cells in the presence of phorbol 12-myristate13-acetate (PMA). Macrophage cells were then treated with different concentrations (10, 20 and 40 mM) of SAC for 24 h. Total RNA of treated macrophages was extracted and analyzed with real-time RT-PCR. ABCA1 protein expression was also analyzed with western blotting. Results showed that SAC increased the ABCA1 mRNA (1.82-, 2.07- and 2.23-fold) and protein (1.37-, 1.55- and 2.08-fold) expression in macrophage THP-1 cells compared with control (untreated cells). Results suggested that SAC can increase ABCA1 expression in macrophages and may be beneficial in promoting reverse cholesterol efflux.