Effects of selected bioactive food compounds on human white adipocyte function

Background: Previous studies suggest that intake of specific bioactive compounds may have beneficial clinical effects on adipose tissue partly due to their anti-inflammatory and insulin-sensitizing properties. With the overall aim to contribute to better understanding of the mechanisms of selected bioactive nutrients on fat metabolism, we investigated their role on human white adipocyte function. Methods: The influence of the omega-3-fatty acid docosahexaenoic acid (DHA), the anthocyanin (AC) cyanidin-3-glucoside and its metabolite protocatechuic acid, and the beta-glucan metabolite propionic acid (PI) on adipokine secretion, fatty acid metabolism (lipolysis/lipogenesis) and adipocyte differentiation (lipid accumulation) was studied in human fat cells differentiated in vitro. To investigate possible synergistic, additive or antagonistic effects, DHA was also combined with AC or PI. Results: Each compound, alone or together with DHA, suppressed basal adipocyte lipolysis compared to control treated cells. DHA alone attenuated the secretion of pro-inflammatory adipokines such as chemerin, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1/CCL2), whereas AC suppressed only the latter two. Treatment with PI decreased IL-6, tumour necrosis factor alpha (TNFα) and adiponectin secretion. A combination of DHA and AC decreased TNFα secretion and increased insulin-stimulated lipogenesis. No effect was found on adipocyte differentiation. At the selected concentrations, none of the compounds was found to be cytotoxic. Conclusion: The studied bioactive food compounds or their metabolites have beneficial effects in human primary fat cells measured as decreased basal lipolytic activity and secretion of inflammatory markers. A minor effect was also observed on insulin-stimulated glucose uptake albeit only with the combination of DHA and AC. Taken together, our results may link the reported health benefits of the selected bioactives on metabolic disorders such as insulin resistance, hypertension and dyslipidemia to effects on white adipocytes

Ceruloplasmin is a novel adipokine which is overexpressed in adipose tissue of obese subjects and in obesity-associated cancer cells

Obesity confers an increased risk of developing specific cancer forms. Although the mechanisms are unclear, increased fat cell secretion of specific proteins (adipokines) may promote/facilitate development of malignant tumors in obesity via cross-talk between adipose tissue(s) and the tissues prone to develop cancer among obese. We searched for novel adipokines that were overexpressed in adipose tissue of obese subjects as well as in tumor cells derived from cancers commonly associated with obesity. For this purpose expression data from human adipose tissue of obese and non-obese as well as from a large panel of human cancer cell lines and corresponding primary cells and tissues were explored. We found expression of ceruloplasmin to be the most enriched in obesity-associated cancer cells. This gene was also significantly up-regulated in adipose tissue of obese subjects. Ceruloplasmin is the body's main copper carrier and is involved in angiogenesis. We demonstrate that ceruloplasmin is a novel adipokine, which is produced and secreted at increased rates in obesity. In the obese state, adipose tissue contributed markedly (up to 22%) to the total circulating protein level. In summary, we have through bioinformatic screening identified ceruloplasmin as a novel adipokine with increased expression in adipose tissue of obese subjects as well as in cells from obesity-associated cancers. Whether there is a causal relationship between adipose overexpression of ceruloplasmin and cancer development in obesity cannot be answered by these cross-sectional comparisons

Role of Receptor-Interacting Protein 140 in human fat cells

<p>Abstract</p> <p>Background</p> <p>Mice lacking <it>Receptor-interacting protein 140 (RIP140) </it>have reduced body fat which at least partly is mediated through increased lipid and glucose metabolism in adipose tissue. In humans, <it>RIP140 </it>is lower expressed in visceral white adipose tissue (WAT) of obese versus lean subjects. We investigated the role of <it>RIP140 </it>in human subcutaneous WAT, which is the major fat depot of the body.</p> <p>Methods</p> <p>Messenger RNA levels of <it>RIP140 </it>were measured in samples of subcutaneous WAT from women with a wide variation in BMI and in different human WAT preparations. <it>RIP140 </it>mRNA was knocked down with siRNA in <it>in vitro </it>differentiated adipocytes and the impact on glucose transport and mRNA levels of target genes determined.</p> <p>Results</p> <p><it>RIP140 </it>mRNA levels in subcutaneous WAT were decreased among obese compared to lean women and increased by weight-loss, but did not associate with mitochondrial DNA copy number. <it>RIP140 </it>expression increased during adipocyte differentiation <it>in vitro </it>and was higher in isolated adipocytes compared to corresponding pieces of WAT. Knock down of <it>RIP140 </it>increased basal glucose transport and mRNA levels of <it>glucose transporter 4 </it>and <it>uncoupling protein-1</it>.</p> <p>Conclusions</p> <p>Human <it>RIP140 </it>inhibits glucose uptake and the expression of genes promoting energy expenditure in the same fashion as the murine orthologue. Increased levels of human <it>RIP140 </it>in subcutaneous WAT of lean subjects may contribute to economize on energy stores. By contrast, the function and expression pattern does not support that <it>RIP140 </it>regulate human obesity.</p

Transforming Growth Factor-β3 Regulates Adipocyte Number in Subcutaneous White Adipose Tissue.

White adipose tissue (WAT) mass is determined by adipocyte size and number. While adipocytes are continuously turned over, the mechanisms controlling fat cell number in WAT upon weight changes are unclear. Herein, prospective studies of human subcutaneous WAT demonstrate that weight gain increases both adipocyte size and number, but the latter remains unaltered after weight loss. Transcriptome analyses associate changes in adipocyte number with the expression of 79 genes. This gene set is enriched for growth factors, out of which one, transforming growth factor-β3 (TGFβ3), stimulates adipocyte progenitor proliferation, resulting in a higher number of cells undergoing differentiation in vitro. The relevance of these observations was corroborated in vivo where Tgfb3+/- mice, in comparison with wild-type littermates, display lower subcutaneous adipocyte progenitor proliferation, WAT hypertrophy, and glucose intolerance. TGFβ3 is therefore a regulator of subcutaneous adipocyte number and may link WAT morphology to glucose metabolism

Regulation of germline transcription in the immunoglobulin heavy chain locus

During an immune response, activated B cells develop into high rate immunoglobulin (Ig) secreting plasma cells. They also switch from production of IgM to IgG, IgA or IgE. Specific isotypes of antibodies have functional and structural features that make them particularly well suited to defend against different types of pathogens. IgM is changed to another isotype via class switch recombination (CSR) - a non-homologous DNA recombination process. CSR is at least in part regulated at the transcriptional level. The external stimuli (cytokines or cell-to-cell contact) induce CSR to a particular constant heavy chain (CH) gene via their ability to modulate germline (GL) transcription of a given CH gene. The GL transcription is driven by GL promoters located upstream of each CH region. The GL promoters contain multiple transcription-factor binding sites and are tightly regulated during B cell differentiation. In addition, several enhancer elements with developmentally regulated activities are located in the IgH locus. The mouse 3' enhancers span a region of more than thirty kilobases and consist of four DNAse hypersensitivity sites - HS3a, HSI,2, HS3b and HS4. One possible function of the 3' enhancers could be regulation of CSR. We aimed to clarify the role of different IgH locus regulatory elements and transcription factors in regulation of GL transcription. Much evidence indicated that T cell-dependent activation of B lymphocytes is generated through the interaction of CD40 (on B cells) with CD40 ligand (on activated T cells). Therefore, using agonistic rat anti-mouse CD40 monoclonal antibody, we investigated the role of CD40 signalling in CSR. We found that stimulation of murine cells through CD40 induced GL y 1, y2b and low levels of e transcripts. However, cells were expressing only IgG2b, but not IgG1 or IgE. CD40, but not lipopolysaccharide (LPS), could induce GL y I transcripts, although both CD40 and LPS signalling involve NF-kB family proteins. We showed that LPS and CD40 stimulation induce different NF-kB complexes binding to GL y I promoter. he increased levels of IgE in atopic individuals could be associated with upregulation of GL e transcription. The transcription factor STAT6 is an important activator of the GL e promoter, induced by interleukin 4 (IL-4). We investigated whether increased levels of IgE in allergic individuals may be associated with alterations in the level or activation of STAT6. Our results demonstrated for the first time that upon IL-4 signalling, STAT6 activation and basal GL E promoter activity differ in B cells from different individuals. Although we did not find any association between STAT6 activation and allergy, we do not exclude the possibility that stronger activation of this transcription factor is associated with the atopic phenotype. Next, we investigated regulation of GL transcription by the IgH locus 3' enhancers. Previous studies have shown that the activity of the HS1,2 enhancer was induced in B cells approximately at the same differentiation stage when GL transcription is activated and CSR occurs. Therefore, we evaluated if HS 1,2 could regulate murine GL e and y2b promoters. We cloned GL promoters with or without HS1,2 in the luciferase expression vector and transiently transfected B cell lines or activated primary B cells. Both GL e and y2b promoters were strongly enhanced by HS1,2 in activated primary B cells. The main activity of the HS1,2 enhancer was found in a fragment being 90% homologous to the human HS1,2. By mutating transcription factor binding sites in HS1,2, we found that NF-AB (Nuclear Factor of Activated B cells) and NF-kB are differently important for interaction between the enhancer and the promoters. Our data imply that specific sites in HSI,2 selectively interact with different GL promoters. We further investigated interaction of the GL e promoter with 3' enhancers in chromatin environment. Transgenic mice containing GL e promoter, switch (Se) region and Cc region were generated. The GL e transgene was expressed in stimulated B cells. The expression was induced by the same stimuli that activated the endogenous GL e promoter (by LPS plus IL-4 or anti-CD40 plus IL-4). This finding indicates that expression of the GL e promoter does not absolutely require presence of the 3' enhancers in the transgenic system. It was shown by others that deletion of HS3a or HSI,2 enhancers has no effect on GL transcription and CSR, while the deletion of HS3bHS4 enhancers causes inhibition of CSR to most isotypes. We aimed at comparing the ability of these enhancerpairs to regulate GL transcription in transgenic mice. Therefore, either HS3a-HSI,2 or HS3b-HS4 were linked to the GL e transgenic construct. The GL e promoter was strongly up-regulated by both parts of the 3' enhancer complex. Expression of the enhancercontaining transgenes was B cell specific. Although, the transgenes were expressed in non-stimulated B cells, the expression could be further enhanced by stimuli. None of the constructs showed properties of a locus control region. We have also analyzed mutation rate in the S region of GL e transgenic mice. These mutations are known to be a sign of recruited recombination machinery. However, we did not find strong evidence for induction of mutations in any of the transgenic constructs, suggesting that other regulatory elements are needed for the recruitment of the recombination machinery

Tables of human adipose tissue cell type-specific genes

The data set includes:<div>1) "Celltype_enriched.txt" files separated by ";" that contain genes significantly enriched in one cell type compared to other three analyzed hWAT cell types according to the analysis described in R markdown file:</div><div>http://figshare.com/s/509ea9441af442a735ac</div><div>Adjusted p-value <0.05 for all pairwise comparisons, moderated F-statistics p-value <0.05 (from R package/bioconductor package Limma).</div><div><br></div><div>2) "Celltype_enriched_collected.txt" files separated by ";". These files contain tables that include mean expression of the gene in a fraction where it is enriched, log fold change (logFC) and adjusted P values (adj.P.val) compared to each of other fractions. Minimal logFC and maximal adj.P.Val against other fractions are included as separate columns to enable easy sorting of the data in search for best expressed, best enriched or most specific genes. </div><div><br></div><div>3) R-code used to generate "Celltype_enriched_collected.txt" tables is provided.</div><div><br></div><div>4) In addition, a table with 100 highest-ranked genes from each fraction (based on highest logFC_min and lowest adj.P.val_max) is also included ("Selected_enriched_genes_all_fractions" - separated by tab).</div><p></p

Mechanism of TNFα-induced downregulation of salt-inducible kinase 2 in adipocytes

Salt-inducible kinase 2 (SIK2) is highly expressed in white adipocytes, but downregulated in individuals with obesity and insulin resistance. These conditions are often associated with a low-grade inflammation in adipose tissue. We and others have previously shown that SIK2 is downregulated by tumor necrosis factor α (TNFα), however, involvement of other pro-inflammatory cytokines, or the mechanisms underlying TNFα-induced SIK2 downregulation, remain to be elucidated. In this study we have shown that TNFα downregulates SIK2 protein expression not only in 3T3L1- but also in human in vitro differentiated adipocytes. Furthermore, monocyte chemoattractant protein-1 and interleukin (IL)-1β, but not IL-6, might also contribute to SIK2 downregulation during inflammation. We observed that TNFα-induced SIK2 downregulation occurred also in the presence of pharmacological inhibitors against several kinases involved in inflammation, namely c-Jun N-terminal kinase, mitogen activated protein kinase kinase 1, p38 mitogen activated protein kinase or inhibitor of nuclear factor kappa-B kinase (IKK). However, IKK may be involved in SIK2 regulation as we detected an increase of SIK2 when inhibiting IKK in the absence of TNFα. Increased knowledge about inflammation-induced downregulation of SIK2 could ultimately be used to develop strategies for the reinstalment of SIK2 expression in insulin resistance

JUP/plakoglobin is regulated by salt-inducible kinase 2, and is required for insulin-induced signalling and glucose uptake in adipocytes

BACKGROUND: Salt-inducible kinase 2 (SIK2) is abundant in adipocytes, but downregulated in adipose tissue from individuals with obesity and insulin resistance. Moreover, SIK isoforms are required for normal insulin signalling and glucose uptake in adipocytes, but the underlying molecular mechanisms are currently not known. The adherens junction protein JUP, also termed plakoglobin or γ-catenin, has recently been reported to promote insulin signalling in muscle cells.OBJECTIVE: The objective of this study was to analyse if JUP is required for insulin signalling in adipocytes and the underlying molecular mechanisms of this regulation.METHODS: Co-expression of SIK2 and JUP mRNA levels in adipose tissue from a human cohort was analysed. siRNA silencing and/or pharmacological inhibition of SIK2, JUP, class IIa HDACs and CRTC2 was employed in 3T3-L1- and primary rat adipocytes. JUP protein expression was analysed by western blot and mRNA levels by qPCR. Insulin signalling was evaluated by western blot as levels of phosphorylated PKB/Akt and AS160, and by monitoring the uptake of 3H-2-deoxyglucose.RESULTS: mRNA expression of SIK2 correlated with that of JUP in human adipose tissue. SIK2 inhibition or silencing resulted in downregulation of JUP mRNA and protein expression in 3T3-L1- and in primary rat adipocytes. Moreover, JUP silencing reduced the expression of PKB and the downstream substrate AS160, and consequently attenuated activity in the insulin signalling pathway, including insulin-induced glucose uptake. The known SIK2 substrates CRTC2 and class IIa HDACs were found to play a role in the SIK-mediated regulation of JUP expression.CONCLUSIONS: These findings identify JUP as a novel player in the regulation of insulin sensitivity in adipocytes, and suggest that changes in JUP expression could contribute to the effect of SIK2 on insulin signalling in these cells