Anna Polus

Increased nitric oxide availability attenuates high fat diet metabolic alterations and gene expression associated with insulin resistance

BackgroundHigh fat diet impairs nitric oxide (NO) bioavailability, and induces insulin resistance. The link between NO availability and the metabolic adaptation to a high fat diet is not well characterized. The purpose of this study was to investigate the effect of high fat diet on metabolism in mice with decreased (eNOS-/-) and increased (DDAH overexpressed) NO bioavailability.MethodseNOS-/- (n = 16), DDAH (n = 24), and WT (n = 19) mice were fed a high fat diet (HFD) for 13 weeks. Body weight, biochemical parameters, adipokines and insulin were monitored. The matrigel in vivo model with CD31 immunostaining was used to assess angiogenesis.Gene expression in adipose tissues was analyzed by microarray and Real Time PCR. Comparisons of the mean values were made using the unpaired Student t test and p < 0.05 were considered statistically significant.ResultseNOS-/- mice gained less weight than control WT and DDAH mice. In DDAH mice, a greater increase in serum adiponectin and a lesser increment in glucose level was observed. Fasting insulin and cholesterol levels remained unchanged. The angiogenic response was increased in DDAH mice. In adipose tissue of DDAH mice, genes characteristic of differentiated adipocytes were down-regulated, whereas in eNOS-/- mice, genes associated with adipogenesis, fatty acid and triglyceride synthesis were upregulated.ConclusionsOur results indicate that increased NO availability attenuates some HFD induced alterations in metabolism and gene expression associated with insulin resistance.

Omega-3 fatty acid supplementation influences the whole blood transcriptome in women with obesity, associated with pro-resolving lipid mediator production.

The n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may reduce low-grade inflammation associated with obesity. The relationship between therapeutic response to n-3 PUFAs and modification of the transcriptome in obesity or metabolic syndrome remains to be explored. Blood samples were obtained from women with obesity before and after three-months supplementation with a moderate dose of n-3 PUFAs (1.8g EPA+DHA per day) or from controls. n-3 PUFAs (GC) and plasma concentrations of lipoxins, resolvins, protectin X (GC-MS/MS) and inflammatory markers (ELISA) were measured. Whole blood transcriptome was assayed using microarray. Women supplemented with n-3 PUFAs for 3months had significantly higher levels of EPA and DHA in plasma phosphatidylcholine. n-3 PUFA supplementation, in contrast to placebo, significantly decreased the concentrations of several inflammatory markers (SELE, MCP-1, sVCAM-1, sPECAM-1, and hsCRP), fasting triglycerides and insulin and increased the concentrations of pro-resolving DHA derivatives in plasma. The microarray data demonstrated effects of n-3 PUFAs on PPAR-α, NRF2 and NF-κB target genes. N-3 PUFAs increased DHA-derived pro-resolving mediators in women with obesity. Elevated resolvins and up-regulation of the resolvin receptor occurred in parallel with activation of PPAR-α target genes related to lipid metabolism and of NRF2 up-regulated antioxidant enzymes.

A Period 2 Genetic Variant Interacts with Plasma SFA to Modify Plasma Lipid Concentrations in Adults with Metabolic Syndrome

Genetic variants of Period 2 (PER2), a circadian clock gene, have been linked to metabolic syndrome (MetS). However, it is still unknown whether these genetic variants interact with the various types of plasma fatty acids. This study investigated whether common single nucleotide polymorphisms (SNPs) in the PER2 locus (rs934945 and rs2304672) interact with various classes of plasma fatty acids to modulate plasma lipid metabolism in 381 participants with MetS in the European LIPGENE study. Interestingly, the rs2304672 SNP interacted with plasma total SFA concentrations to affect fasting plasma TG, TG-rich lipoprotein (TRL-TG), total cholesterol, apoC-II, apoB, and apoB-48 concentrations (P-interaction < 0.001-0.046). Carriers of the minor allele (GC+GG) with the highest SFA concentration (>median) had a higher plasma TG concentration (P = 0.001) and higher TRL-TG (P < 0.001) than the CC genotype. In addition, participants carrying the minor G allele for rs2304672 SNP and with a higher SFA concentration (>median) had higher plasma concentrations of apo C-II (P < 0.001), apo C-III (P = 0.009), and apoB-48 (P = 0.028) compared with the homozygotes for the major allele (CC). In summary, the rs2304672 polymorphism in the PER2 gene locus may influence lipid metabolism by interacting with the plasma total SFA concentration in participants with MetS. The understanding of these gene-nutrient interactions could help to provide a better knowledge of the pathogenesis in MetS.

Angiogenesis in the New Zealand obese mouse model fed with high fat diet

BackgroundObesity and its complications lead to vascular injury, atherosclerosis, diabetes and pathological angiogenesis. One of the models to study the obesity and its entanglements is the New Zealand Obese mice model. Aim of this study was to check the effect of high fat diet on changes in biochemical parameters as well as on process of angiogenesis in NZO mice.MethodsNZO mice were fed with standard (ST) or high fat (HF) diet for seven weeks. Body weight and serum biochemical parameters were monitored. The PECAM1 positive vessel-like structures immunostaining, as well as the gene expression of the matrigel penetrating cells by microarray (confirmed by real-time PCR method) were analyzed.ResultsMice fed with HF diet developed obesity. Number of newly created vessels with lumen was correlated with hyperglycemia and animal weight gain. The number of PECAM1 positive cells in matrigel tended to increase during HF diet. Microarray results revealed changes in gene expression (activation of the oxidative stress and insulin resistance, inhibition of apoptosis and cell differentiation), however without markers of endothelial cell network maturation.ConclusionObserved changes in the NZO mice on HF diet argue for the hyperglycemia related activation of angiogenesis, leading to the formation of pathological, immature network.

β-Carotene stimulates chemotaxis of human endothelial progenitor cells

Abstract Angiogenesis is a crucial process in tissue remodeling during growth, both in the embryo and the adult. In our study we concentrated on the direct effect of β-carotene on human umbilical cord originating from endothelial progenitor cells (EPCs). β-Carotene uptake by EPCs was measured using a HPLC method. The determination of cell surface antigens was performed by flow cytometry. The effect on cell proliferation was estimated by measuring bromo-deoxyuridine incorporation. The influence on the formation of a tubular-like structure was investigated in a 3D assay in matrigel. Quantitative gene expression was estimated using real-time PCR. We demonstrated that β-carotene in the physiological range of concentrations found in human blood is a potent activator of EPC chemotaxis, which is accompanied by a change in the expression of genes mediating cell adhesion and homing, but does not activate the final markers of endothelial differentiation. This study points to the prochemotactic and homing activity of β-carotene in undifferentiated endothelial cell progenitors for the first time, which may suggest a potential role of this carotenoid in progenitor cell therapy aimed at angiogenesis and tissue repair.

The Microarray Expression Analysis Identifies BAX as a Mediator of β-Carotene Effects on Apoptosis

Abstract: β-Carotene is a ubiquitous compound rich in foods. However, there are conflicting reports regarding its role in carcinogenesis. We performed a microarray expression analysis in normal [human umbilical vein endothelial cells (HUVECs)] and neoplastic (melanoma A375 and myelomonocytic leukemia U937) actively proliferating cells and found evidence that β-carotene stimulated vital cellular functions in the former and suppressed them in the latter. These differential effects correlated with the expression of the proapoptotic BCL2-associated X protein (BAX), which was downregulated in HUVECs and upregulated in the two neoplastic cell lines. The quantitative expression analysis using real-time polymerase chain reaction largely confirmed the inhibition of B-cell CLL/lymphoma 2 (BCL2) pathway-mediated apoptosis in HUVECs and its activation in melanoma and leukemic cells. The assays for apoptosis, detecting DNA breaks and caspase activation, showed consistent proapoptotic and antiapoptotic effects in U937 and HUVEC lines, respectively. However, β-carotene–induced expression changes of BAX and other BCL2 pathway genes did not lead to the predicted induction of apoptosis in the A375 cells.

Metabolic effects of the HIV protease inhibitor – saquinavir in differentiating human preadipocytes

BACKGROUND The iatrogenic, HIV-related lipodystrophy is associated with development of the significant metabolic and cardiovascular complications. The underlying mechanisms of antiretroviral (ARV) drugs are not completely explored. METHODS The aim of the study was to characterize effects of the protease inhibitor (PI)--saquinavir (SQV) on metabolic functions, and gene expression during differentiation in cells (Chub-S7) culture. RESULTS SQV in concentrations observed during antiretroviral therapy (ART) significantly decreased mitochondrial membrane potential (MMP), oxygen consumption and ATP generation. The effects were greater in already differentiated cells. This was accompanied by characteristic changes in the expression of the genes involved in endoplasmic reticulum (ER) stress, and differentiation (lipid droplet formation) process such as: WNT10a, C/EBPa, AFT4, CIDEC, ADIPOQ, LPIN1. CONCLUSIONS The results indicate that SQV affects not only metabolic (mitochondrial) activity of adipocytes, but affects the expression of genes related to differentiation and to a lesser extent to cell apoptosis.

Influence of dietary fatty acids on differentiation of human stromal vascular fraction preadipocytes.

Mediators such as cytokines, eicosanoids, nitric oxide and growth factors may regulate adipogenesis as well as inflammation. It is well documented that production of some form of eicosanoids activates lipid synthesis during adipogenesis but also contributes to the formation of factors maintaining low-level systemic inflammation. Developing nutrients for reduction of adipogenesis and inflammation can enhance preventive efficacy of daily diet. This study examined the effects of free fatty acid influence on changes in lipid biosynthesis and corresponding gene expression during differentiation of human subcutaneous adipose tissue stromal vascular fraction (SVF) cells. Proadipogenic conditions promoted SVF cell differentiation and lipid droplet (LD) formation up to 15 days. This correlated with gene expression of adipocyte differentiation markers as well as inflammatory cytokines and their receptors. Addition of free fatty acids to differentiation medium increased their incorporation during the first period of differentiation (48 h). Presence of eicosanoid acid (EPA) during the initial period of differentiation by elevation of Perilipin 3 protein (TIP47), may be responsible for smaller LD formation. Presence of arachidonic acid (AA) tends to deposit lipids in large form of LDs. Prolongation of differentiation up to 15 days decreased AA or EPA in cellular lipids. PUFA through up-regulation of both phospholipase 2 and enzymes related to eicosanoid production influenced type and quantity of eicosanoids which regulated the extent of SVF cell differentiation. Formation of small LDs and reduction of pro-inflammatory mediators in adipose tissue are the consequence of eicosanoid production with anti-inflammatory potential from EPA.

Effect of caloric restriction with or without n-3 polyunsaturated fatty acids on insulin sensitivity in obese subjects: a randomized placebo controlled trial

Background Caloric restriction and n-3 polyunsaturated fatty acid (PUFA) supplementation protect from some of the metabolic complications. The aim of this study was to assess the influence of a low calorie diet with or without n-3 PUFA supplementation on glucose dependent insulinotropic polypeptide (GIP) output and insulin sensitivity markers in obese subjects. Methods Obese, non-diabetic subjects (BMI 30–40 kg/m2) and aged 25–65 yr. were put on low calorie diet (1200–1500 kcal/day) supplemented with either 1.8 g/day n-3 PUFA (DHA/EPA, 5:1) (n = 24) or placebo capsules (n = 24) for three months in a randomized placebo controlled trial. Insulin resistance markers and GIP levels were analysed from samples obtained at fasting and during an oral glucose tolerance test (OGTT). Results Caloric restriction with n-3 PUFA led to a decrease of insulin resistance index (HOMA-IR) and a significant reduction of insulin output as well as decreased GIP secretion during the OGTT. These effects were not seen with caloric restriction alone. Changes in GIP output were inversely associated with changes in red blood cell EPA content whereas fasting GIP level positively correlated with HOMA-IR index. Blood triglyceride level was lowered by caloric restriction with a greater effect when n-3 PUFA were included and correlated positively with fasting GIP level. Conclusions Three months of caloric restriction with DHA + EPA supplementation exerts beneficial effects on insulin resistance, GIP and triglycerides. General significance Combining caloric restriction and n-3 PUFA improves insulin sensitivity, which may be related to a decrease of GIP levels.

Differentiation of human adipose tissue SVF cells into cardiomyocytes

Progenitor cells have been extensively studied and therapeutically applied in tissue reconstructive therapy. Stromal vascular fraction (SVF) cells, which are derived from adipose tissue, may represent a potential source of the cells which undergo phenotypical differentiation into many lineages both in vitro as well as in vivo. The goal of this study was to check whether human SVF cells may differentiate into cardiomyocyte-like entities. Human SVF cells were induced to differentiate by their incubation in Methocult medium in the presence of SCF, IL-3 and IL-6. Morphological transformation of the cells was monitored using optical light microscope, whereas changes in expression of the genes typical for cardiac phenotype were measured by qRT-PCR. Incubation of the human SVF cells in the medium that promotes cardiomyocyte differentiation in vitro resulted in formation of myotubule-like structures accompanied by up-regulation of the myocardium-characteristic genes, such as GATA, MEF2C, MYOD1, but not ANP. Human SVF cells differentiate into cardiomyocyte-like cells in the presence of the certain set of myogenesis promoting cytokines.