Jana Kračmerová

Weight Loss Improves the Adipogenic Capacity of Human Preadipocytes and Modulates Their Secretory Profile

Calorie restriction–induced weight loss is accompanied by profound changes in adipose tissue characteristics. To determine the effect of weight loss on differentiation of preadipocytes and secretory capacity of in vitro differentiated adipocytes, we established cultures of these cells from paired subcutaneous adipose tissue biopsies obtained before and at the end of weight-reducing dietary intervention (DI) in 23 obese women. Based on lipid accumulation and the expression of differentiation markers, in vitro adipogenesis increased after weight loss and it was accompanied by enhanced expression of genes involved in de novo lipogenesis. This effect of weight loss was not driven by changes of peroxisome proliferator–activated receptor γ sensitivity to rosiglitazone. Weight loss also enhanced the expression of adiponectin and leptin while reducing that of monocyte chemoattractant protein 1 and interleukin-8 by cultured adipocytes. Thus, the weight-reducing (DI) increased adipogenic capacity of preadipocytes and shifted their secretion toward lower inflammatory profile. Reprogramming of preadipocytes could represent an adaptation to weight loss leading to partial restoration of preobese adipose tissue traits and thus contribute to the improvement of metabolic status. However, enhanced adipogenesis could also contribute to the unwanted weight regain after initial weight loss.

Soluble CD163 Is Associated With CD163 mRNA Expression in Adipose Tissue and With Insulin Sensitivity in Steady-State Condition but Not in Response to Calorie Restriction

CONTEXT Soluble CD163 (sCD163) was suggested as a biomarker of insulin sensitivity and CD163 mRNA expression representing macrophage content in adipose tissue (AT). OBJECTIVE The aim of this study was to investigate, in cross-sectional and prospective design, the relationship between sCD163 circulating levels and CD163 mRNA expression in adipose tissue and insulin sensitivity assessed by euglycemic-hyperinsulinemic clamp. DESIGN, SETTING, PARTICIPANTS, AND INTERVENTIONS Two cohorts of subjects were examined in the study. Cohort 1 included 42 women with a wide range of body mass index (17-48 kg/m(2)); cohort 2 included 27 obese women who followed a dietary intervention consisting of 1 month of a very low-calorie diet and 5 months of a weight-stabilization period. MAIN OUTCOME MEASURES Serum levels of CD163 and mRNA expression of CD163 and CD68 in sc and visceral (visc) AT were determined, and insulin sensitivity [expressed as glucose disposal rate (GDR)] was measured in cohort 1. In cohort 2, serum levels of CD163, mRNA expressions of CD163, CD68, and CD163-shedding factors [TNF-α-converting enzyme (TACE) and tissue inhibitor of metalloproteinase (TIMP3)] in sc AT were examined and GDR was measured before and during dietary intervention. RESULTS In cohort 1, circulating sCD163 correlated with CD163 mRNA levels in both sc and visc AT. sCD163 and CD163 mRNA expression in both fat depots correlated with GDR. In cohort 2, the diet-induced changes of sCD163 levels did not correlate with those of CD163, CD68, TACE, and TIMP3 mRNA levels. Although the pattern of the diet-induced change of sCD163 paralleled that of GDR, there was no correlation between the changes of these two variables. CONCLUSION sCD163 correlates with CD163 mRNA expression in sc and visc AT and with whole-body insulin sensitivity in the steady-state condition. These associations are not observed with respect to the diet-induced changes during a weight-reducing hypocaloric diet.

Ursodeoxycholic Acid but Not Tauroursodeoxycholic Acid Inhibits Proliferation and Differentiation of Human Subcutaneous Adipocytes

Stress of endoplasmic reticulum (ERS) is one of the molecular triggers of adipocyte dysfunction and chronic low inflammation accompanying obesity. ERS can be alleviated by chemical chaperones from the family of bile acids (BAs). Thus, two BAs currently used to treat cholestasis, ursodeoxycholic and tauroursodeoxycholic acid (UDCA and TUDCA), could potentially lessen adverse metabolic effects of obesity. Nevertheless, BAs effects on human adipose cells are mostly unknown. They could regulate gene expression through pathways different from their chaperone function, namely through activation of farnesoid X receptor (FXR) and TGR5, G-coupled receptor. Therefore, this study aimed to analyze effects of UDCA and TUDCA on human preadipocytes and differentiated adipocytes derived from paired samples of two distinct subcutaneous adipose tissue depots, abdominal and gluteal. While TUDCA did not alter proliferation of cells from either depot, UDCA exerted strong anti-proliferative effect. In differentiated adipocytes, acute exposition to neither TUDCA nor UDCA was able to reduce effect of ERS stressor tunicamycin. However, exposure of cells to UDCA during whole differentiation process decreased expression of ERS markers. At the same time however, UDCA profoundly inhibited adipogenic conversion of cells. UDCA abolished expression of PPARγ and lipogenic enzymes already in the early phases of adipogenesis. This anti-adipogenic effect of UDCA was not dependent on FXR or TGR5 activation, but could be related to ability of UDCA to sustain the activation of ERK1/2 previously linked with PPARγ inactivation. Finally, neither BAs did lower expression of chemokines inducible by TLR4 pathway, when UDCA enhanced their expression in gluteal adipocytes. Therefore while TUDCA has neutral effect on human preadipocytes and adipocytes, the therapeutic use of UDCA different from treating cholestatic diseases should be considered with caution because UDCA alters functions of human adipose cells.

Experimental hyperglycemia induces an increase of monocyte and T-lymphocyte content in adipose tissue of healthy obese women.

Background/Objectives Hyperglycemia represents one of possible mediators for activation of immune system and may contribute to worsening of inflammatory state associated with obesity. The aim of our study was to investigate the effect of a short-term hyperglycemia (HG) on the phenotype and relative content of immune cells in circulation and subcutaneous abdominal adipose tissue (SAAT) in obese women without metabolic complications. Subjects/Methods Three hour HG clamp with infusion of octreotide and control investigations with infusion of octreotide or saline were performed in three groups of obese women (Group1: HG, Group 2: Octreotide, Group 3: Saline, n=10 per group). Before and at the end of the interventions, samples of SAAT and blood were obtained. The relative content of immune cells in blood and SAAT was determined by flow cytometry. Gene expression analysis of immunity-related markers in SAAT was performed by quantitative real-time PCR. Results In blood, no changes in analysed immune cell population were observed in response to HG. In SAAT, HG induced an increase in the content of CD206 negative monocytes/macrophages (p<0.05) and T lymphocytes (both T helper and T cytotoxic lymphocytes, p<0.01). Further, HG promoted an increase of mRNA levels of immune response markers (CCL2, TLR4, TNFα) and lymphocyte markers (CD3g, CD4, CD8a, TBX21, GATA3, FoxP3) in SAAT (p<0.05 and 0.01). Under both control infusions, none of these changes were observed. Conclusions Acute HG significantly increased the content of monocytes and lymphocytes in SAAT of healthy obese women. This result suggests that the short-term HG can modulate an immune status of AT in obese subjects.

The antimicrobial action of histones in the reproductive tract of cow.

An infection of any part of female reproductive tract can severely interfere with fertility and reproduction. The fluids and epithelium from the lumen of the female reproductive tract (uterus, oviduct and ovarian follicle) are a known source of antimicrobial action in several species. In this study, we compared the antimicrobial properties of fluids from the reproductive tract of a cow. After removal of small molecules, we demonstrated that there is an antimicrobial activity connected with a fraction of compounds with a molecular mass range between 3500 and 30,000. The most probable candidates responsible for the observed antimicrobial effect were subsequently identified by mass spectroscopy as histones H2A type 2-C, H2B type 1-K, H3.3, and H4. The antimicrobial role of histone H2B was further confirmed by using an antibody against this histone.

Native Red Electrophoresis – A new method suitable for separation of native proteins

A new type of native electrophoresis was developed to separate and characterize proteins. In this modification of the native blue electrophoresis, the dye Ponceau Red S is used instead of Coomassie Brilliant Blue to impose uniform negative charge on proteins to enable their electrophoretic separation according to their relative molecular masses. As Ponceau Red S binds less tightly to proteins, in comparison with Coomassie Blue, it can be easily removed after the electrophoretic separation and a further investigation of protein properties is made possible (e.g. an enzyme detection or electroblotting). The tested proteins also kept their native properties (enzyme activity or aggregation state).

Stress of endoplasmic reticulum modulates differentiation and lipogenesis of human adipocytes.

BACKGROUND Adipocytes are cells specialized for storage of neutral lipids. This storage capacity is dependent on lipogenesis and is diminished in obesity. The reason for the decline in lipogenic activity of adipocytes in obesity remains unknown. Recent data show that lipogenesis in liver is regulated by pathways initiated by endoplasmic reticulum stress (ERS). Thus, we aimed at investigating the effect of ERS on lipogenesis in adipose cells. METHODS Preadipocytes were isolated from subcutaneous abdominal adipose tissue from obese volunteers and in vitro differentiated into adipocytes. ERS was induced pharmacologically by thapsigargin (TG) or tunicamycin (TM). Activation of Unfolded Protein Response pathway (UPR) was monitored on the level of eIF2α phosphorylation and mRNA expression of downstream targets of UPR sensors. Adipogenic and lipogenic capacity was evaluated by Oil Red O staining, measurement of incorporation of radio-labelled glucose or acetic acid into lipids and mRNA analysis of adipogenic/lipogenic markers. RESULTS Exposition of adipocytes to high doses of TG (100 nM) and TM (1 μg/ml) for 1-24 h enhanced expression of several UPR markers (HSPA5, EDEM1, ATF4, XBP1s) and phosphorylation of eIF2α. This acute ERS substantially inhibited expression of lipogenic genes (DGAT2, FASN, SCD1) and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to low dose of TG (2.5 nM) during the early phases of adipogenic conversion of preadipocytes impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in mature adipocytes. CONCLUSIONS Acute ERS weakened a capacity of mature adipocytes to store lipids and chronic ERS diminished adipogenic potential of preadipocytes.

Postprandial inflammation is not associated with endoplasmic reticulum stress in peripheral blood mononuclear cells from healthy lean men.

The consumption of lipids and simple sugars induces an inflammatory response whose exact molecular trigger remains elusive. The aims of the present study were to investigate (1) whether inflammation induced by a single high-energy, high-fat meal (HFM) is associated with endoplasmic reticulum stress (ERS) in peripheral blood mononuclear cells (PBMC) and (2) whether these inflammatory and ERS responses could be prevented by the chemical chaperone ursodeoxycholic acid (UDCA). A total of ten healthy lean men were recruited to a randomised, blind, cross-over trial. Subjects were given two doses of placebo (lactose) or UDCA before the consumption of a HFM (6151 kJ; 47·4 % lipids). Blood was collected at baseline and 4 h after the HFM challenge. Cell populations and their activation were analysed using flow cytometry, and plasma levels of inflammatory cytokines were assessed by ELISA and Luminex technology. Gene expression levels of inflammatory and ERS markers were analysed in CD14⁺ and CD14⁻ PBMC using quantitative RT-PCR. The HFM induced an increase in the mRNA expression levels of pro-inflammatory cytokines (IL-1β, 2·1-fold; IL-8, 2·4-fold; TNF-α, 1·4-fold; monocyte chemoattractant protein 1, 2·1-fold) and a decrease in the expression levels of miR181 (0·8-fold) in CD14⁺ monocytes. The HFM challenge did not up-regulate the expression of ERS markers (XBP1, HSPA5, EDEM1, DNAJC3 and ATF4) in either CD14⁺ or CD14⁻ cell populations, except for ATF3 (2·3-fold). The administration of UDCA before the consumption of the HFM did not alter the HFM-induced change in the expression levels of ERS or inflammatory markers. In conclusion, HFM-induced inflammation detectable on the level of gene expression in PBMC was not associated with the concomitant increase in the expression levels of ERS markers and could not be prevented by UDCA.

Native polyacrylamide electrophoresis in the presence of Ponceau Red to study oligomeric states of protein complexes

Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.