Toll-like receptor 2 and Toll-like receptor 4 expression in human adrenals.

Toll-like receptors (TLRs) are key elements in the innate immune response, functioning as pattern-recognition receptors for the detection and response to endotoxins and other microbial ligands. Inflammatory cytokines play an important role in the activation of the hypothalamic-pituitary-adrenal HPA axis during inflammation and sepsis. The newly recognized major role of TLR2 and TLR4 and the adrenal stress response during critical illnesses such as inflammation and sepsis demand comprehensive analysis of their interactions. Therefore, we analyzed TLR2 and TLR4 expression in human adrenal glands. Western blot analysis demonstrated the expression of TLR2 and TLR4 in the human adrenocortical cell line NCI-H295. Immunohistochemical analysis of normal human adrenal glands revealed TLR2 and TLR4 expression in the adrenal cortex, but not in the adrenal medulla. Considering the crucial role of the HPA axis and the innate immune response during acute sepsis or septic shock, elucidating the functional interaction of these systems should be of great clinical relevance.     

Comparison of the expression and activity of the lipogenic pathway in human and rat adipose tissue

Lipogenesis is considered less active in human than in rat adipose tissue. This could be explained by different nutritional conditions, namely high-carbohydrate (HCHO) diet in rats and high-fat (HF) diet in humans. Adipose tissue was sampled (postabsorptive state) in rats and humans receiving HCHO or HF diets, ad libitum fed humans, and obese subjects. We measured 1) mRNA concentrations of fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), sterol regulatory element binding protein 1c (SREBP-1c), and carbohydrate response element binding protein (ChREBP), 2) SREBP-1c protein, and 3) FAS activity. FAS, ACC1, ChREBP, and SREBP1-c mRNA concentrations were unaffected by diet in humans or in rats. FAS and ACC1 mRNA levels were lower in humans than in rats (P < 0.05). FAS activity was unaffected by diet and was lower in humans (P < 0.05). SREBP-1c mRNA concentrations were similar in rats and humans, but the precursor and mature forms of SREBP-1c protein were less abundant in humans (P < 0.05). ChREBP mRNA concentrations were lower in humans than in rats. In conclusion, the lipogenic capacity of adipose tissue is lower in humans than in rats. This is not related to differences in diet and is probably explained by lower abundance of SREBP-1c protein. A decreased expression of ChREBP could also play a role.     

Toll-like receptor family and signalling pathway

Toll is a Drosophila gene essential for ontogenesis and anti-microbial resistance. Several orthologues of Toll have been identified and cloned in vertebrates, namely Toll-like receptors (TLRs). Human TLRs are a growing family of molecules involved in innate immunity. TLRs are characterized structurally by a cytoplasmic Toll/interleukin-1 receptor (TIR) domain and by extracellular leucine-rich repeats. TLRs characterized so far activate the MyD88/interleukin-1 receptor-associated kinase (IRAK) signalling pathway. Genetic, gene-transfer and dominant-negative approaches have involved TLR family members (TLR2 and TLR4) in Gram-positive and Gram-negative bacteria recognition and signalling. Accumulating evidence suggests that TLR2 is also involved in signalling-receptor complexes that recognize components of yeast and mycobacteria. However, the definitive roles of other TLRs are still lacking. A systematic approach has been used to determine whether different human leucocyte populations selectively or specifically express TLR mRNA. Based on expression pattern, TLR can be classified as ubiquitous (TLR1), restricted (TLR2, TLR4 and TLR5) and specific (TLR3). Expression and regulation of distinct but overlapping ligand-recognition patterns may underlie the existence of a large, seemingly redundant TLR family. Alternatively, the expression of a TLR in a single cell type may indicate a specific role for this molecule in a restricted setting.     

Role of Toll-like receptor 4/NF-kappaB pathway in monocyte-endothelial adhesion induced by low shear stress and ox-LDL

TLR4 plays an important role in atherosclerosis, but little is known about the precise mechanism. Herein, we investigated the role of TLR4/NF-kappaB signaling pathway in monocyte-endothelial adhesion induced by low shear stress and Ox-LDL. We found that low shear stress up-regulated TLR4 expression in endothelial cells, and that ox-LDL exerted an obvious synergistic action as revealed by RT-PCR and Western blotting analysis. Low shear stress also significantly up-regulated IL-8 expression in endothelial cells. Meanwhile, NF-kappaB activity and the adhesion force of monocytes were increased, and there was a synergetic action of ox-LDL. However, following transfection with a functional mutant of TLR4 (C3H/HeJ, TLR4 Dicd) or addition of anti-human TLR4 mAb, IL-8 expression was obviously decreased, NF-kappaB activity in cells remarkably inhibited, and the adhesion force of monocyte significantly reduced. Nevertheless, anti-human TLR2 mAb had no similar effects. These findings suggest that TLR4 may be involved in the early stages of atherosclerosis, associating ox-LDL, inflammation/infection, and low shear stress. Therefore, TLR4 is expected to be a new target for preventing and treating atherosclerosis.     

Prolactin and growth hormone regulate adiponectin secretion and receptor expression in adipose tissue

Adiponectin is a hormone secreted from adipose tissue, and serum levels are decreased with obesity and insulin resistance. Because prolactin (PRL) and growth hormone (GH) can affect insulin sensitivity, we investigated the effects of these hormones on the regulation of adiponectin in human adipose tissue in vitro and in rodents in vivo. Adiponectin secretion was significantly suppressed by PRL and GH in in vitro cultured human adipose tissue. Furthermore, PRL increased adiponectin receptor 1 (AdipoR1) mRNA expression and GH decreased AdipoR2 expression in the cultured human adipose tissue. In transgenic mice expressing GH, and female mice expressing PRL, serum levels of adiponectin were decreased. In contrast, GH receptor deficient mice had elevated adiponectin levels, while PRL receptor deficient mice were unaffected. In conclusion, we demonstrate gene expression of AdipoR1 and AdipoR2 in human adipose tissue for the first time, and show that these are differentially regulated by PRL and GH. Both PRL and GH reduced adiponectin secretion in human adipose tissue in vitro and in mice in vivo. Decreased serum adiponectin levels have been associated with insulin resistance, and our data in human tissue and in transgenic mice suggest a role for adiponectin in PRL and GH induced insulin resistance.     

Characterization of microRNA expression in bovine adipose tissues: a potential regulatory mechanism of subcutaneous adipose tissue development

Background  

MicroRNAs (miRNAs), a family of small non-coding RNA molecules, appear to regulate animal lipid metabolism and preadipocyte conversion to form lipid-assimilating adipocytes (i.e. adipogenesis). However, no miRNA to date has been reported to modulate adipogenesis and lipid deposition in beef cattle.     

Heterogeneous expression of Toll-like receptor 4 and downregulation of Toll-like receptor 4 expression on human gingival fibroblasts by Porphyromonas gingivalis lipopolysaccharide.

Porphyromonas gingivalis (P. gingivalis) is implicated in the initiation and progression of periodontitis. Human gingival fibroblasts (HGFs) are the major constituent of gingival connective tissue. P. gingivalis or its components such as lipopolysaccharide (LPS) upregulate the production of various inflammatory cytokines including interleukin (IL)-1 and IL-6 in HGFs. Recently, we demonstrated that the binding of P. gingivalis LPS to Toll-like receptor 4 (TLR4) on HGFs activates various second messenger systems (Biochem. Biophys. Res. Commun. 273, 1161-1167, 2000). In the present study, we examined the level of TLR4 expression on HGFs by flow cytometric analysis (FACS), and studied the levels of IL-1 and IL-6 in the culture medium upon LPS stimulation of HGFs by enzyme-linked immunosorbent assay (ELISA). Upon stimulation by P. gingivalis LPS for 24 h, HGFs that expressed a high level of TLR4 secreted significantly higher levels of IL-1 and IL-6 than HGFs that expressed a low level of TLR4. On the other hand, after stimulation with P. gingivalis LPS for 24 h, the level of TLR4 on the surface of HGFs decreased. These results suggest that the level of TLR4 expression on HGFs reflects the extent of inflammation in the gingival tissue, and that P. gingivalis LPS downregulates TLR4 expression on HGFs. These findings may be used to control inflammatory and immune responses in periodontal disease.     

ITIH-5 expression in human adipose tissue is increased in obesity

Adipocytes secrete many proteins that regulate metabolic functions. The gene inter-α (globulin) inhibitor H5 (ITIH-5) encodes a secreted protein and is known to be expressed abundantly in the placenta. However, using gene expression profiles data we observed high expression of ITIH-5 in adipose tissue. The aim of this study was to test the hypothesis that ITIH-5 is strongly expressed in human adipocytes and adipose tissue, and is related to obesity and clinical metabolic variables. ITIH-5 adipose tissue mRNA expression was analyzed with DNA microarray and real-time PCR, and its association with clinical variables was examined. ITIH-5 protein expression was analyzed using western blot. ITIH-5 mRNA expression was abundant in human adipose tissue, adipocytes, and placenta, and higher in subcutaneous (sc) compared to omental adipose tissue (P < 0.0001). ITIH-5 mRNA and protein expression in sc adipose tissue were higher in obese compared to lean subjects (P < 0.0001 and P < 0.001, respectively). ITIH-5 mRNA expression was reduced after diet-induced weight loss (P < 0.0001). ITIH-5 mRNA expression was associated with anthropometry and clinical metabolic variables. In conclusion, ITIH-5 is highly expressed in sc adipose tissue, increased in obesity, down regulated after weight loss, and associated with measures of body size and metabolism. Together, this indicates that ITIH-5 merits further investigation as a regulator of human metabolism.     

IMPDHII protein inhibits Toll-like receptor 2-mediated activation of NF-kappaB

Toll-like receptor 2 (TLR2) plays an essential role in innate immunity by the recognition of a large variety of pathogen-associated molecular patterns. It induces its recruitment to lipid rafts induces the formation of a membranous activation cluster necessary to enhance, amplify, and control downstream signaling. However, the exact composition of the TLR2-mediated molecular complex is unknown. We performed a proteomic analysis in lipopeptide-stimulated THP1 and found IMPDHII protein rapidly recruited to lipid raft. Whereas IMPDHII is essential for lymphocyte proliferation, its biologic function within innate immune signal pathways has not been established yet. We report here that IMPDHII plays an important role in the negative regulation of TLR2 signaling by modulating PI3K activity. Indeed, IMPDHII increases the phosphatase activity of SHP1, which participates to the inactivation of PI3K.     

Negative regulation of Toll-like receptor signaling pathway

TLRs are primary sensors of invading pathogens, recognizing conserved microbial molecules and activating signaling pathways that are pivotal to innate and adaptive immune responses. However, a TLR signaling pathway must be tightly controlled because its excessive activation can contribute to the pathogenesis of many human diseases. This review provides a summary of the different mechanisms that are involved in the negative regulation of TLR signaling pathways.     

Heterogeneity of human liver, muscle, and adipose tissue insulin receptor

We have studied the structure and function of the human insulin receptor in liver, skeletal muscle and adipose tissue. The alpha-subunit of the insulin receptor for liver, muscle and adipose tissue migrated on SDS-PAGE with Mrs 137632 +/- 216, 134034 +/- 1080, and 133575 +/- 165, respectively (p less than 0.05). Treatment of these receptors with neuraminidase decreased their molecule sizes and eliminated the relative size differences between the receptors. Three monoclonal antibodies (5A1, 10D9, and 20H3), directed towards different epitopes of the human insulin receptor alpha-subunit were used to probe immunological differences among the receptors. Antibodies 5A1 and 20H3 recognized all the receptors, whereas 10D9 recognized muscle and adipose tissue receptors but not liver receptors. The mobility of insulin receptor beta-subunit in the absence of insulin was the same in all tissues with a similar phosphorylation-induced decrease in mobility in SDS-PAGE in the presence of insulin. However, insulin stimulated autophosphorylation per receptor was different being greatest (p less than 0.05) in muscle (334 +/- 104 32P cpm) and similar in adipose tissue (114 +/- 10) and liver (183 +/- 68). These studies indicate, therefore, that the human insulin receptor is heterogeneous among the major target tissues for insulin, and raise the possibility that this heterogeneity may account for tissues' specific differences in insulin's biological messages.     

NF-kappaB regulates the expression of the human complement receptor 2 gene

CR2 is a key regulator of the B cell response to Ag. Here we show that NF-kappaB enhances the expression of the human CR2 gene. Promoter truncation, deletion, and mutagenesis studies indicated a functional role for a consensus NF-kappaB promoter element, as well as a heterogeneous nuclear ribonucleoprotein D element and an overlapping X box/E box. By supershift analysis, the first two elements bound NF-kappaB p50 and p65 and heterogeneous nuclear ribonucleoprotein RNP D, respectively. The X box/E box bound regulatory factor X5 and, surprisingly, NF-kappaB p50 and p65. Overexpression of NF-kappaB p50 enhanced the activity of the CR2 promoter in B cell lines and primary B cells, suggesting a direct role for NF-kappaB in regulating promoter activity. Importantly, mutation of the NF-kappaB element or the X box/E box rendered the promoter unresponsive to NF-kappaB p50. Using chromatin immunoprecipitation in live B cell lines and primary B cells, we found that NF-kappaB proteins p50, p65, and c-Rel bound to the genomic promoter at two locations that overlap with the consensus NF-kappaB element or the X box/E box. Finally, stimuli that activate NF-kappaB enhanced the activity of the CR2 promoter, and LPS rapidly increased the number of CR2 proteins on the surface of primary B cells. We propose that the NF-kappaB signaling pathway enhances the expression of the CR2 gene, as a result of NF-kappaB proteins binding to two CR2 promoter elements. Thus, at the onset of an infection, LPS could sensitize the B cell to Ag by enhancing the level of CR2-costimulatory molecules on the cell surface.     

Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-kappa B activation

Toll-like receptor (TLR) 4 has been identified as the primary receptor for enteric LPS, whereas TLR2 has been implicated as the receptor for Gram-positive and fungal cell wall components and for bacterial, mycobacterial, and spirochetal lipoproteins. Vascular endothelial cell (EC) activation or injury by microbial cell wall components such as LPS is of critical importance in the development of sepsis and septic shock. We have previously shown that EC express predominantly TLR4, and have very little TLR2. These cells respond vigorously to LPS via TLR4, but are unresponsive to lipoproteins and other TLR2 ligands. Here we show that LPS, TNF-alpha, or IFN-gamma induce TLR2 expression in both human dermal microvessel EC and HUVEC. Furthermore, LPS and IFN-gamma act synergistically to induce TLR2 expression in EC, and LPS-induced TLR2 expression is NF-kappaB dependent. LPS and IFN-gamma also up-regulate TLR4 mRNA expression in EC. These data indicate that TLR2 and TLR4 expression in ECs is regulated by inflammatory molecules such as LPS, TNF-alpha, or IFN-gamma. TLR2 and TLR4 molecules may render EC responsive to TLR2 ligands and may help to explain the synergy between LPS and lipoproteins, and between LPS and IFN-gamma, in inducing shock associated with Gram-negative sepsis.     

Metallothionein gene expression in human adipose tissue from lean and obese subjects.

Expression of the gene encoding metallothionein, a low molecular-weight cysteine-rich, stress-response and metal-binding protein was examined in human adipose tissue. The mRNA for MT-2A, a major metallothionein isoform in humans, was detected in subcutaneous fat using a specific antisense oligonucleotide probe. The level of MT-2A mRNA was significantly higher in a group of obese subjects than in a lean group, paralleling a similar increase in ob mRNA. A two-week period on a diet of 800 calories/day did not lead to any significant change in MT-2 mRNA levels. Separation of mature adipocytes from the cells of the stromal vascular fraction indicated that in human adipose tissue the metallothionein (MT-2A) gene is expressed both in adipocytes and in other cells of the tissue.     

Analysis of an expression profile of genes in the human adipose tissue

Increasing evidence suggests that in addition to storing excess energy as fat, adipose tissue acts as an endocrine organ secreting various factors into the blood stream. Every time a new factor is found in adipose tissue, however, its implication is discussed independently, and a systematic analyses based upon a global view of gene expression of this tissue has not been performed. To describe the function of this tissue in terms of gene expression, and to find new factors, we performed random complementary DNA (cDNA) sequencing using a 3'-directed cDNA library that faithfully represents the composition of the messenger RNA (mRNA). Various well-known but unexpected genes, including those for gelsolin, plasma glutathione peroxidase (GPX-3) and carboxypeptidase E (CPE) were shown to be very active. By comparing the expression profile of active genes in the adipose with those of other tissues and with data in dbEST, we identified seven new genes that are specifically expressed in adipose tissue. Among these, one encoded a protein with collagen-like repeats and a putative secretion signal. These data can be used as new tools for analyses of the physiology of this tissue, as well as the etiology and complications of obesity.     

Insulin signalling in human adipose tissue

Adipose tissue is a critical regulator of energy balance and substrate metabolism, and synthesizes several different substances with endocrine or paracrine functions, which regulate the overall energetic homeostasis. An excessive amount of adipose tissue has been associated with the development of type 2 diabetes, premature atherosclerosis, and cardiovascular disease. It is believed that the adverse metabolic impact of visceral fat relies on a relative resistance to the action of insulin in this depot compared to other adipose tissue depots. However, information on insulin signalling reactions in human fat is limited. In this paper, we review the major insulin signalling pathways in adipocytes and their relevance for metabolic regulation, and discuss recent data indicating different signalling properties of visceral fat as compared to other fat depots, which may explain the metabolic and hormonal specificity of this fat tissue depot in humans.