Agné Kulyté

Adipose Tissue MicroRNAs as Regulators of CCL2 Production in Human Obesity

In obesity, white adipose tissue (WAT) inflammation is linked to insulin resistance. Increased adipocyte chemokine (C-C motif) ligand 2 (CCL2) secretion may initiate adipose inflammation by attracting the migration of inflammatory cells into the tissue. Using an unbiased approach, we identified adipose microRNAs (miRNAs) that are dysregulated in human obesity and assessed their possible role in controlling CCL2 production. In subcutaneous WAT obtained from 56 subjects, 11 miRNAs were present in all subjects and downregulated in obesity. Of these, 10 affected adipocyte CCL2 secretion in vitro and for 2 miRNAs (miR-126 and miR-193b), regulatory circuits were defined. While miR-126 bound directly to the 3′-untranslated region of CCL2 mRNA, miR-193b regulated CCL2 production indirectly through a network of transcription factors, many of which have been identified in other inflammatory conditions. In addition, overexpression of miR-193b and miR-126 in a human monocyte/macrophage cell line attenuated CCL2 production. The levels of the two miRNAs in subcutaneous WAT were significantly associated with CCL2 secretion (miR-193b) and expression of integrin, α-X, an inflammatory macrophage marker (miR-193b and miR-126). Taken together, our data suggest that miRNAs may be important regulators of adipose inflammation through their effects on CCL2 release from human adipocytes and macrophages.

MicroRNA regulatory networks in human adipose tissue and obesity

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and, therefore, biological processes in different tissues. A major function of miRNAs in adipose tissue is to stimulate or inhibit the differentiation of adipocytes, and to regulate specific metabolic and endocrine functions. Numerous miRNAs are present in human adipose tissue; however, the expression of only a few is altered in individuals with obesity and type 2 diabetes mellitus or are differentially expressed in various adipose depots. In humans, obesity is associated with chronic low-grade inflammation that is regulated by signal transduction networks, in which miRNAs, either directly or indirectly (through regulatory elements such as transcription factors), influence the expression and secretion of inflammatory proteins. In addition to their diverse effects on signalling, miRNAs and transcription factors can interact to amplify the inflammatory effect. Although additional miRNA signal networks in human adipose tissue are not yet known, similar regulatory circuits have been described in brown adipose tissue in mice. miRNAs can also be secreted from fat cells into the circulation and serve as markers of disturbed adipose tissue function. Given their role in regulating transcriptional networks, miRNAs in adipose tissue might offer tangible targets for treating metabolic disorders.

Adipose tissue pathways involved in weight loss of cancer cachexia

Background:The regulatory gene pathways that accompany loss of adipose tissue in cancer cachexia are unknown and were explored using pangenomic transcriptome profiling.Methods:Global gene expression profiles of abdominal subcutaneous adipose tissue were studied in gastrointestinal cancer patients with (n=13) or without (n=14) cachexia.Results:Cachexia was accompanied by preferential loss of adipose tissue and decreased fat cell volume, but not number. Adipose tissue pathways regulating energy turnover were upregulated, whereas genes in pathways related to cell and tissue structure (cellular adhesion, extracellular matrix and actin cytoskeleton) were downregulated in cachectic patients. Transcriptional response elements for hepatic nuclear factor-4 (HNF4) were overrepresented in the promoters of extracellular matrix and adhesion molecule genes, and adipose HNF4 mRNA was downregulated in cachexia.Conclusions:Cancer cachexia is characterised by preferential loss of adipose tissue; muscle mass is less affected. Loss of adipose tissue is secondary to a decrease in adipocyte lipid content and associates with changes in the expression of genes that regulate energy turnover, cytoskeleton and extracellular matrix, which suggest high tissue remodelling. Changes in gene expression in cachexia are reciprocal to those observed in obesity, suggesting that regulation of fat mass at least partly corresponds to two sides of the same coin.

MicroRNAs Regulate Human Adipocyte Lipolysis: Effects of miR-145 Are Linked to TNF-α

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d) modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.

Inhibition of Mycobacterium smegmatis Gene Expression and Growth Using Antisense Peptide Nucleic Acids

Antisense agents that inhibit genes at the mRNA level are attractive tools for genome-wide studies and drug target validation. The approach may be particularly well suited to studies of bacteria that are difficult to manipulate with standard genetic tools. Antisense peptide nucleic acids (PNA) with attached carrier peptides can inhibit gene expression in Escherichia coli and Staphylococcus aureus. Here we asked whether peptide-PNAs could mediate antisense effects in Mycobacterium smegmatis. We first targeted the gfp reporter gene and observed dose- and sequence-dependent inhibition at low micromolar concentrations. Sequence alterations within both the PNA and target mRNA sequences eliminated inhibition, strongly supporting an antisense mechanism of inhibition. Also, antisense PNAs with various attached peptides showed improved anti-gfp effects. Two peptide-PNAs targeted to the essential gene inhA were growth inhibitory and caused cell morphology changes that resemble that of InhA-depleted cells. Therefore, antisense peptide-PNAs can efficiently and specifically inhibit both reporter and endogenous essential genes in mycobacteria.

MicroRNA profiling links miR-378 to enhanced adipocyte lipolysis in human cancer cachexia

Cancer cachexia is associated with pronounced adipose tissue loss due to, at least in part, increased fat cell lipolysis. MicroRNAs (miRNAs) have recently been implicated in controlling several aspects of adipocyte function. To gain insight into the possible impact of miRNAs on adipose lipolysis in cancer cachexia, global miRNA expression was explored in abdominal subcutaneous adipose tissue from gastrointestinal cancer patients with (n = 10) or without (n = 11) cachexia. Effects of miRNA overexpression or inhibition on lipolysis were determined in human in vitro differentiated adipocytes. Out of 116 miRNAs present in adipose tissue, five displayed distinct cachexia-associated expression according to both microarray and RT-qPCR. Four (miR-483-5p/-23a/-744/-99b) were downregulated, whereas one (miR-378) was significantly upregulated in cachexia. Adipose expression of miR-378 associated strongly and positively with catecholamine-stimulated lipolysis in adipocytes. This correlation is most probably causal because overexpression of miR-378 in human adipocytes increased catecholamine-stimulated lipolysis. In addition, inhibition of miR-378 expression attenuated stimulated lipolysis and reduced the expression of LIPE, PLIN1, and PNPLA2, a set of genes encoding key lipolytic regulators. Taken together, increased miR-378 expression could play an etiological role in cancer cachexia-associated adipose tissue loss via effects on adipocyte lipolysis.

Additive Effects of MicroRNAs and Transcription Factors on CCL2 Production in Human White Adipose Tissue

Adipose tissue inflammation is present in insulin-resistant conditions. We recently proposed a network of microRNAs (miRNAs) and transcription factors (TFs) regulating the production of the proinflammatory chemokine (C-C motif) ligand-2 (CCL2) in adipose tissue. We presently extended and further validated this network and investigated if the circuits controlling CCL2 can interact in human adipocytes and macrophages. The updated subnetwork predicted that miR-126/-193b/-92a control CCL2 production by several TFs, including v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) (ETS1), MYC-associated factor X (MAX), and specificity protein 12 (SP1). This was confirmed in human adipocytes by the observation that gene silencing of ETS1, MAX, or SP1 attenuated CCL2 production. Combined gene silencing of ETS1 and MAX resulted in an additive reduction in CCL2 production. Moreover, overexpression of miR-126/-193b/-92a in different pairwise combinations reduced CCL2 secretion more efficiently than either miRNA alone. However, although effects on CCL2 secretion by co-overexpression of miR-92a/-193b and miR-92a/-126 were additive in adipocytes, the combination of miR-126/-193b was primarily additive in macrophages. Signals for miR-92a and -193b converged on the nuclear factor-κB pathway. In conclusion, TF and miRNA-mediated regulation of CCL2 production is additive and partly relayed by cell-specific networks in human adipose tissue that may be important for the development of insulin resistance/type 2 diabetes.

An AMP-activated protein kinase-stabilizing peptide ameliorates adipose tissue wasting in cancer cachexia in mice

Cachexia represents a fatal energy-wasting syndrome in a large number of patients with cancer that mostly results in a pathological loss of skeletal muscle and adipose tissue. Here we show that tumor cell exposure and tumor growth in mice triggered a futile energy-wasting cycle in cultured white adipocytes and white adipose tissue (WAT), respectively. Although uncoupling protein 1 (Ucp1)-dependent thermogenesis was dispensable for tumor-induced body wasting, WAT from cachectic mice and tumor-cell-supernatant-treated adipocytes were consistently characterized by the simultaneous induction of both lipolytic and lipogenic pathways. Paradoxically, this was accompanied by an inactivated AMP-activated protein kinase (Ampk), which is normally activated in peripheral tissues during states of low cellular energy. Ampk inactivation correlated with its degradation and with upregulation of the Ampk-interacting protein Cidea. Therefore, we developed an Ampk-stabilizing peptide, ACIP, which was able to ameliorate WAT wasting in vitro and in vivo by shielding the Cidea-targeted interaction surface on Ampk. Thus, our data establish the Ucp1-independent remodeling of adipocyte lipid homeostasis as a key event in tumor-induced WAT wasting, and we propose the ACIP-dependent preservation of Ampk integrity in the WAT as a concept in future therapies for cachexia.

Variable coordination of cotranscribed genes in Escherichia coli following antisense repression

BackgroundA majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target.ResultsTo examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment.ConclusionThe results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria.

MTCH2 in Human White Adipose Tissue and Obesity

CONTEXT Genome-wide association studies have identified single-nucleotide polymorphisms in approximately 40 loci associated with obesity-related traits. How these loci regulate obesity is largely unknown. One obesity-associated single-nucleotide polymorphism is close to the MTCH2 gene (mitochondrial carrier homolog 2). OBJECTIVE The objective of the study was to assess the expression of genes in obesity-associated loci in abdominal sc white adipose tissue (scWAT) in relation to obesity. A more comprehensive expression study was performed on MTCH2. DESIGN mRNA levels of 66 genes from 40 loci were determined by microarray in scWAT from lean and obese women (n = 30). MTCH2 mRNA was measured by quantitative RT-PCR in lean and obese before and after weight loss in intact adipose pieces and isolated adipocytes, paired samples of scWAT and omental WAT, and primary adipocyte cultures (n = 191 subjects in total). MTCH2 genotypes were compared with mRNA expression in 96 women. MTCH2 protein was examined in scWAT of 38 individuals. RESULTS Adipose expression of eight genes was significantly associated with obesity; of these, MTCH2 displayed the highest absolute signal. MTCH2 mRNA and protein expression was significantly increased in obese women but was not affected by weight loss. MTCH2 was enriched in isolated fat cells and increased during adipocyte differentiation. There was no cis influence of MTCH2 genotypes on mRNA levels. CONCLUSION MTCH2 is highly expressed in human WAT and adipocytes with increased levels in obese women. These results suggest that MTCH2 may play a role in cellular processes underlying obesity.