Propionic acid counteracts the inflammation of human subcutaneous adipose tissue: a new avenue for drug development

Adipose tissue is a primary site of obesity-induced inflammation, which has been emerging as an important contributor to obesity associated disorders. The factors influencing adipose tissue-induced inflammation and the resulting pathophysiological events remain poorly understood. However, dietary fiber consumptions appear to be protective. Short-chain fatty acids such as propionic acid (PA) are the principal products of the dietary fiber fermentation by microbiota. Therefore, we aim to investigate the influence of PA on inflammation, lipogenesis and glucose uptake markers from human subcutaneous adipose tissue (SAT). We showed that the treatment of SAT with PA resulted in a significant downregulation of inflammatory parameters (e.g. TNF-α and IP-10) and macrophage markers (e.g. CD163 and MMP-9). The expression levels of PA receptors (i.e. G protein coupled receptor-41 and -43) in human primary adipocytes were very low in comparison with SAT and macrophages. Upon PA treatment, no anti-inflammatory effect was observed in human adipocytes. PA significantly upregulated the expression of lipoprotein lipase (LPL), sterol regulatory-element-binding protein-1c (SREBP-1c) and glucose transporter 4 (GLUT-4), which are associated with lipogenesis and glucose uptake. We also showed that the observed anti-inflammatory effects of PA on SAT were partly mediated by Gi/o protein coupled receptor. Our data suggests that PA anti-inflammatory effects on SAT are mediated partly via Gi/o proteins, leading to the improved expression of factors associated with lipogenesis and glucose uptake. These responses appeared to be not mediated by adipocytes; but most probably by macrophages. The current study provides new knowledge, which can be used as a potential new avenue for drug development in preventing obesity-related inflammation and metabolic disorders in future. [Figure not available: see fulltext.]

Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3′ and 5′ terminals of targets leads to the self-assembly of dsDNAs into the sulfur- rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 × 10-9 mol.L-1 over a linear ranged from 20 × 10-9 to 10 × 10-7 mol.L-1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications

Human Primary Adipocytes Exhibit Immune Cell Function: Adipocytes Prime Inflammation Independent of Macrophages

BACKGROUND: Obesity promotes inflammation in adipose tissue (AT) and this is implicated in pathophysiological complications such as insulin resistance, type 2 diabetes and cardiovascular disease. Although based on the classical hypothesis, necrotic AT adipocytes (ATA) in obese state activate AT macrophages (ATM) that then lead to a sustained chronic inflammation in AT, the link between human adipocytes and the source of inflammation in AT has not been in-depth and systematically studied. So we decided as a new hypothesis to investigate human primary adipocytes alone to see whether they are able to prime inflammation in AT. METHODS AND RESULTS: Using mRNA expression, human preadipocytes and adipocytes express the cytokines/chemokines and their receptors, MHC II molecule genes and 14 acute phase reactants including C-reactive protein. Using multiplex ELISA revealed the expression of 50 cytokine/chemokine proteins by human adipocytes. Upon lipopolysaccharide stimulation, most of these adipocyte-associated cytokines/chemokines and immune cell modulating receptors were up-regulated and a few down-regulated such as (ICAM-1, VCAM-1, MCP-1, IP-10, IL-6, IL-8, TNF-α and TNF-β highly up-regulated and IL-2, IL-7, IL-10, IL-13 and VEGF down-regulated. In migration assay, human adipocyte-derived chemokines attracted significantly more CD4+ T cells than controls and the number of migrated CD4+ cells was doubled after treating the adipocytes with LPS. Neutralizing MCP-1 effect produced by adipocytes reduced CD4+ migration by approximately 30%. CONCLUSION: Human adipocytes express many cytokines/chemokines that are biologically functional. They are able to induce inflammation and activate CD4+ cells independent of macrophages. This suggests that the primary event in the sequence leading to chronic inflammation in AT is metabolic dysfunction in adipocytes, followed by production of immunological mediators by these adipocytes, which is then exacerbated by activated ATM, activation and recruitment of immune cells. This study provides novel knowledge about the prime of inflammation in human obese adipose tissue, opening a new avenue of investigations towards obesity-associated type 2 diabetes

Repression of Smoothened by Patched-Dependent (Pro-)Vitamin D3 Secretion

The developmentally important hedgehog (Hh) pathway is activated by binding of Hh to patched (Ptch1), releasing smoothened (Smo) and the downstream transcription factor glioma associated (Gli) from inhibition. The mechanism behind Ptch1-dependent Smo inhibition remains unresolved. We now show that by mixing Ptch1-transfected and Ptch1 small interfering RNA–transfected cells with Gli reporter cells, Ptch1 is capable of non–cell autonomous repression of Smo. The magnitude of this non–cell autonomous repression of Smo activity was comparable to the fusion of Ptch1-transfected cell lines and Gli reporter cell lines, suggesting that it is the predominant mode of action. CHOD-PAP analysis of medium conditioned by Ptch1-transfected cells showed an elevated 3β-hydroxysteroid content, which we hypothesized to mediate the Smo inhibition. Indeed, the inhibition of 3β-hydroxysteroid synthesis impaired Ptch1 action on Smo, whereas adding the 3β-hydroxysteroid (pro-)vitamin D3 to the medium effectively inhibited Gli activity. Vitamin D3 bound to Smo with high affinity in a cyclopamine-sensitive manner. Treating zebrafish embryos with vitamin D3 mimicked the smo (–/–) phenotype, confirming the inhibitory action in vivo. Hh activates its signalling cascade by inhibiting Ptch1-dependent secretion of the 3β-hydroxysteroid (pro-)vitamin D3. This action not only explains the seemingly contradictory cause of Smith-Lemli-Opitz syndrome (SLOS), but also establishes Hh as a unique morphogen, because binding of Hh on one cell is capable of activating Hh-dependent signalling cascades on other cells

Differentially Modulated Ptch1 Action in <i>Dhcr7 ^–/–</i> and <i>Sc5d ^–/–</i> MEFs

<div><p>(A) Basal Gli reporter activity measured in MEFs either deficient for Sc5d or Dhcr7 shows that the stacking of 7-DHC (in the <i>Dhcr7^–/–</i> MEFs) lowers Gli activity in the mutant cells compared with lathosterol stacking in the <i>Sc5d^–/–</i> MEFs. Transfer of the conditioned media to reporter cells (indicated as “MEFs as medium donors”) shows that the inhibitory potential of the <i>Dhcr7^–/–</i> MEFs is medium-borne. Media were incubated on donor MEFs for 16 h and transferred to reporter cells for 6 to 8 h. ( <i>n</i> ≥ 4; *, <i>p</i> < 0.05; ***, <i>p</i> < 0.005). </p> <p>(B) Medium transfer of Ptch1, Ptch1 siRNA, or control-transfected <i>Sc5d^–/–</i> and <i>Dhcr7^–/–</i> MEFs shows that cells stacking lathosterol but lacking 7-DHC ( <i>Sc5d^–/–</i> MEFs) are not able to translate different Ptch1 levels to an inhibitory action on reporter cells. The <i>Dhcr7^–/–</i> cells were able to properly convey an inhibitory signal when transfected with Ptch1 or, inversely, show a diminished inhibitory potential upon <i>Ptch1</i> siRNA transfection. UVB treatment of Ptch1 transfectant medium (2 h) amplified the inhibitory action. Media incubations and statistics are as in (A). </p></div

Cholesterol Synthesis and Possible Modes of Action of Ptch1

<div><p>(A) Shown is a schematic representation of cholesterol synthesis. Boxed are lathosterol and 7-DHC, cholesterol precursors known to accumulate in lathosterolosis and SLOS patients, respectively. Mutations in or genetic loss of sterol C5-desaturase <i>(Sc5d)</i> cause stacking of lathosterol, whereas dysfunction of 7-DHC reductase <i>(Dchr7)</i> or the addition of its synthetic inhibitor AY-9944 causes accumulation of 7-DHC. The conversion of 7-DHC to vitamin D3 (cholecalciferol) is mediated by UV light. Statins, like pravastatin, inhibit HMG-CoA reductase, the enzyme that forms mevalonate. </p> <p>(B) Shown are three possible models for the inhibitory action of the Hh receptor Ptch1 on Smo. (1) A cell-autonomous mode of action, in which direct binding of Ptch1 inhibits Smo. (2) An intracellular inhibitory action, mediated by direct binding of Ptch1 to Smo. (3) The model in which Ptch1 pumps an inhibitory small molecule that is capable of Smo-repression intercellularly (as well as intracellularly; not shown).</p></div

Effects of Vitamin D3 on Developing Zebrafish Embryos

<div><p>(A) Fertilized zebrafish eggs were dechorionated and placed in buffer containing 1.2 mg/ml sonicated vitamin D3, starting the continuous incubation between the 32- and 64-cell stages. At the different developmental stages (as indicated), the embryos were analysed, revealing persistent somite malformations, a ventrally curved body, poorly developed myoseptum, an aberrant extension of the yolk tube, a prominent dorsal midbrain, and a reduced trunk. The wild-type Engrailed staining (4D9) of muscle pioneers and surrounding cells in control-treated animals was not observed upon vitamin D3 treatment. ISH for <i>ptc1</i> mRNA showed a strongly reduced signal in vitamin D3–treated animals compared with controls. Staining with the F59 antibody revealed that slow muscle fibres were disturbed in number and orientation in the treated animals. </p> <p>(B) At 18 hpf, embryos were photographed in detail and somite angles were determined (as shown in top panel). The measurement was taken of an individual somite between dorsal and ventral portions of the vertical myoseptum for corresponding somites between the wild-type and vitamin D3–treated embryos. Shown are the mean angles (±SEM; <i>n</i> = 3) of wild-type control- and vitamin D3–treated embryos. </p> <p>C) Detail of 4-dpf embryo. Note the slightly enlarged pericardial cavity in vitamin D3–treated animals. The orientation of all images is a lateral view, anterior to the left.</p></div

Confirmation of Functionality of Constructs and Model System Used

<div><p>(A) Transfection of Ptch1 expression construct in C3H/10T1/2 fibroblasts increased Ptch1 expression over basal expression, as seen on Western blot. Actin levels remained unaltered; cells were lysed 24 h post transfection.</p> <p>(B) C3H/10T1/2 cells are sensitive to Hh pathway components as indicated by Gli reporter activity when pathway components are expressed: Smo increased Hh pathway activity as determined by Gli reporter luciferase assay. Cotransfection of Ptch1 suppressed Smo-induced Gli activation. Transfection of Ptch1 in the absence of Smo overexpression did not decrease Gli activity below control levels. Shh stimulation (1 μg/ml for 6 h, 16 h post transfection) and transfection of a Gli1 expression construct showed highest reporter activity, as expected. Addition of 1 μg/ml Shh-blocking antibody 5E1 reduced Shh-mediated activation of Gli reporter activity. The Ptch1-insensitive mutant SmoM2 showed a high basal activity that could not be diminished by cotransfecting Ptch1, as expected. Cells were transfected and lysed after 24 h. Values shown are relative light unit (RLU) values corrected for an internal CMV Renilla standard, expressed as percent increase relative to vector (pcDNA 3.1–) transfection or control stimulation. Depicted is the mean ± SEM. ( <i>n</i> = 4; *, <i>p</i> < 0.05; **, <i>p</i> < 0.01). </p> <p>(C) Shh concentration in medium is below the detection limit (5 ng/ml) of Western blotting. Medium was spiked with decreasing concentrations of recombinant Shh, and blotted along with a 4× concentrated medium sample obtained from C3H/10T1/2 fibroblasts (incubated for 16 h at a volume-to-surface ratio identical to the mix-and-match and medium transfer experiments).</p></div