Administration of Nicotine Exacerbates the Quinine-induced Structural and Functional Alterations of Testicular Tissue in Adult Rats: An Experimental Study.

PURPOSE: In this study the role of nicotine (NCT) administration on the intensity of rat testicular tissue alterations induced by quinine (QU) was evaluated. MATERIALS AND METHODS: Forty adult Wistar rats were divided into four groups. Control (CON), NCT administrated (4 mg/kg) (NCT), QU treated (25 mg/kg for 7 days) (QU), and nicotine with quinine received (NCT+QU). After 28 days, serum testosterone and malondialdehyde (MDA) levels were measured. Testes and epididymides samples were prepared for determining tissue MDA levels, histomorphometry, microscopic indices of spermatogenesis, immunohistochemistry of p53 and sperm analysis. RESULTS: Testosterone levels were decreased significantly (P = .0004) in treated groups compared to CON group. Serum MDA levels were increased significantly (P = .0004) in NCT and QU groups compared to CON group. Tissue MDA levels were increased significantly (P = .0012) in NCT+QU group in comparison to CON group. These parameters were changed significantly in NCT+QU group compared to QU group. Seminiferous tubules diameter decreased significantly (P < .0001) in treated groups compared to CON group and in NCT+QU group compared to QU group. The height of germinal epithelium decreased significantly (P = .0001) in NCT and NCT+QU groups compared to CON and QU groups. The number of Sertoli cells, spermatocytes, and spermatids decreased significantly in treated groups compared to CON group. The number of spermatogonia decreased significantly (P = .0017) in NCT and NCT+QU groups compared to CON group. The number of Sertoli cells, spermatogonia, and spermatocytes decreased significantly in NCT+QU group compared to QU group. All indices of spermatogenesis decreased in treated groups compared to CON group. The lowest mean of these indices was observed in NCT+QU group. The sperm viability decreased significantly (P < .0001) in treated groups compared to CON group. Sperm count and motility decreased significantly in NCT and NCT+QU groups compared to CON group. All experimental groups showed the over-expression of p53 compared to CON group. CONCLUSION: The administration of nicotine could be involved in the exacerbation of testicular tissue alterations related to quinine therapy.

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. Graphical abstract Schematic presentation of study flow and the components of the investigation. In this study the effect of propionic acid (PA) on inflammation investigated in human subcutaneous adipose tissue (SAT), human primary adipocytes and the expression of a few hallmark inflammatory components produced by SAT and human adipocytes.

Regulation of Macrophage Recognition through the Interplay of Nanoparticle Surface Functionality and Protein Corona.

Using a family of cationic gold nanoparticles (NPs) with similar size and charge, we demonstrate that proper surface engineering can control the nature and identity of protein corona in physiological serum conditions. The protein coronas were highly dependent on the hydrophobicity and arrangement of chemical motifs on NP surface. The NPs were uptaken in macrophages in a corona-dependent manner, predominantly through recognition of specific complement proteins in the NP corona. Taken together, this study shows that surface functionality can be used to tune the protein corona formed on NP surface, dictating the interaction of NPs with macrophages.

DNA Hypomethylation and Histone Variant macroH2A1 Synergistically Attenuate Chemotherapy-Induced Senescence to Promote Hepatocellular Carcinoma Progression.

Aging is a major risk factor for progression of liver diseases to hepatocellular carcinoma (HCC). Cellular senescence contributes to age-related tissue dysfunction, but the epigenetic basis underlying drug-induced senescence remains unclear. macroH2A1, a variant of histone H2A, is a marker of senescence-associated heterochromatic foci that synergizes with DNA methylation to silence tumor-suppressor genes in human fibroblasts. In this study, we investigated the relationship between macroH2A1 splice variants, macroH2A1.1 and macroH2A1.2, and liver carcinogenesis. We found that protein levels of both macroH2A1 isoforms were increased in the livers of very elderly rodents and humans, and were robust immunohistochemical markers of human cirrhosis and HCC. In response to the chemotherapeutic and DNA-demethylating agent 5-aza-deoxycytidine (5-aza-dC), transgenic expression of macroH2A1 isoforms in HCC cell lines prevented the emergence of a senescent-like phenotype and induced synergistic global DNA hypomethylation. Conversely, macroH2A1 depletion amplified the antiproliferative effects of 5-aza-dC in HCC cells, but failed to enhance senescence. Senescence-associated secretory phenotype and whole-transcriptome analyses implicated the p38 MAPK/IL8 pathway in mediating macroH2A1-dependent escape of HCC cells from chemotherapy-induced senescence. Furthermore, chromatin immunoprecipitation sequencing revealed that this hepatic antisenescence state also required active transcription that could not be attributed to genomic occupancy of these histones. Collectively, our findings reveal a new mechanism by which drug-induced senescence is epigenetically regulated by macroH2A1 and DNA methylation and suggest macroH2A1 as a novel biomarker of hepatic senescence that could potentially predict prognosis and disease progression.

Protease activated receptor-1 regulates macrophage-mediated cellular senescence: a risk for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a destructive disease in part resulting from premature or mature cellular aging. Protease-activated receptor-1 (PAR-1) recently emerged as a critical component in the context of fibrotic lung diseases. Therefore, we aimed to study the role of macrophages in PAR-1-mediated idiopathic pulmonary fibrosis. The number of macrophages were significantly reduced in lungs of PAR-1 antagonist (P1pal-12) treated animals upon bleomycin instillation. In line with these data, PAR-1 stimulation increased monocyte / macrophage recruitment in response to epithelium injury in in vitro trans-well assays. Moreover, macrophages induced fibroblasts migration, differentiation and secretion of collagen, which were inhibited in the presence of TGF-ß receptor inhibitors. Interestingly, these profibrotic effects were partially inhibited by treatment with the PAR-1 inhibitor P1pal-12. Using shRNA mediated PAR-1 knock down in fibroblasts, we demonstrate that fibroblast PAR-1 contributes to TGF-ß activation and production. Finally, we show that the macrophage-dependent induction of PAR-1 driven TGF-ß activation was mediated by FXa. Our data identify novel mechanisms by which PAR-1 stimulation on different cell types can contribute to IPF and identify macrophages as key players in PAR-1 dependent development of this devastating disease. IPF may result from cellular senescence mediated by macrophages in the lung.

Age-related obesity and type 2 diabetes dysregulate neuronal associated genes and proteins in humans.

Despite numerous developed drugs based on glucose metabolism interventions for treatment of age-related diseases such as diabetes neuropathies (DNs), DNs are still increasing in patients with type 1 or type 2 diabetes (T1D, T2D). We aimed to identify novel candidates in adipose tissue (AT) and pancreas with T2D for targeting to develop new drugs for DNs therapy. AT-T2D displayed 15 (e.g. SYT4 up-regulated and VGF down-regulated) and pancreas-T2D showed 10 (e.g. BAG3 up-regulated, VAV3 and APOA1 down-regulated) highly differentially expressed genes with neuronal functions as compared to control tissues. ELISA was blindly performed to measure proteins of 5 most differentially expressed genes in 41 human subjects. SYT4 protein was upregulated, VAV3 and APOA1 were down-regulated, and BAG3 remained unchanged in 1- Obese and 2- Obese-T2D without insulin, VGF protein was higher in these two groups as well as in group 3- Obese-T2D receiving insulin than 4-lean subjects. Interaction networks analysis of these 5 genes showed several metabolic pathways (e.g. lipid metabolism and insulin signaling). Pancreas is a novel site for APOA1 synthesis. VGF is synthesized in AT and could be considered as good diagnostic, and even prognostic, marker for age-induced diseases obesity and T2D. This study provides new targets for rational drugs development for the therapy of age-related DNs.

Significance of surface charge and shell material of superparamagnetic iron oxide nanoparticle (SPION) based core/shell nanoparticles on the composition of the protein corona.

As nanoparticles (NPs) are increasingly used in many applications their safety and efficient applications in nanomedicine have become concerns. Protein coronas on nanomaterials' surfaces can influence how the cell "recognizes" nanoparticles, as well as the in vitro and in vivo NPs' behaviors. The SuperParamagnetic Iron Oxide Nanoparticle (SPION) is one of the most prominent agents because of its superparamagnetic properties, which is useful for separation applications. To mimic surface properties of different types of NPs, a core-shell SPION library was prepared by coating with different surfaces: polyvinyl alcohol polymer (PVA) (positive, neutral and negative), SiO2 (positive and negative), titanium dioxide and metal gold. The SPIONs with different surfaces were incubated at a fixed serum : nanoparticle surface ratio, magnetically trapped and washed. The tightly bound proteins were quantified and identified. The surface charge has a great impact on protein adsorption, especially on PVA and silica where proteins preferred binding to the neutral and positively charged surfaces. The importance of surface material on protein adsorption was also revealed by preferential binding on TiO2 and gold coated SPION, even negatively charged. There is no correlation between the protein net charge and the nanoparticle surface charge on protein binding, nor direct correlation between the serum proteins' concentration and the proteins detected in the coronas.

Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes.

Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic ß-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health.

Human monocytes produce interferon-gamma upon stimulation with LPS.

Representing a crucial T-helper 1 cytokine, IFN-γ acts as an important bridge between innate and adaptive immunity and is involved in many acute and chronic pathologic states, such as autoimmune diseases and solid organ transplant rejection. At present, debate still prevails about the ability of human monocytes to produce IFN-γ. We aimed to investigate whether human monocytes possess the capacity to produce IFN-γ at mRNA and protein level. Using real time PCR, flow cytometric analysis and ELISA, we investigated the capacity of freshly isolated CD14+ monocytes of healthy individuals and kidney transplant recipients to produce IFN-γ after stimulation with IFN-γ and LPS or LPS alone. We observed increased IFN-γ mRNA levels in CD14+ monocytes after stimulation as compared to the unstimulated controls in both populations. In addition, stimulation with IFN-γ and LPS or LPS alone led to a significant increase in the percentage of CD14+ monocytes producing TNF-α and IFN-γ at protein level (p<0.05). A trend towards increased secreted IFN-γ production in supernatants was also observed after LPS stimulation using ELISA. We conclude that human monocytes from healthy individuals and kidney transplant recipients possess the capacity to produce IFN-γ.

Personalized protein coronas: a "key" factor at the nanobiointerface.

It is now well known that the primary interactions of biological entities (e.g., tissues and cells) with nanoparticles (NPs) are strongly influenced by the protein composition of the "corona" (i.e., the NP surface attached proteins). The composition of the corona strongly depends on the protein source (e.g., human plasma). Because the protein source determines the NP corona, it is reasonable to hypothesize that humans with specific disease(s) may have specific NP coronas. To test this hypothesis, we incubated two different hydrophobic/hydrophilic types of NPs (polystyrene and silica) with plasma from human subjects with different diseases and medical conditions (e.g., breast cancer, diabetes, hypercholesterolemia, rheumatism, fauvism, smoking, hemodialysis, thalassemia, hemophilia A and B, pregnancy, common cold and hypofibrinogenemia). Our results demonstrate that the type of disease has a crucial role in the protein composition of the NP corona. Based on these results, we introduce the concept of the "personalized protein corona" (PPC) as a determinant factor in nano-biomedical science. This study will help researchers rationally design experiments based on the "personalized protein corona" for clinical and biological applications.

A shift towards pro-inflammatory CD16+ monocyte subsets with preserved cytokine production potential after kidney transplantation.

BACKGROUND: The presence of monocyte-macrophage lineage cells in rejecting kidney transplants is associated with worse graft outcome. At present, it is still unclear how the monocyte-macrophage related responses develop after transplantation. Here, we studied the dynamics, phenotypic and functional characteristics of circulating monocytes during the first 6 months after transplantation and aimed to establish the differences between kidney transplant recipients and healthy individuals. METHODS: Phenotype, activation status and cytokine production capacity of classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++), monocytes were determined by flow cytometry in a cohort of 33 healthy individuals, 30 renal transplant recipients at transplantation, 19 recipients at 3 months and 16 recipients at 6 months after transplantation using a cross-sectional approach. RESULTS: The percentage of both CD16+ monocyte subsets was significantly increased in transplant recipients compared to healthy individuals, indicative of triggered innate immunity (p≤0.039). Enhanced production capacity of tumor necrosis factor-α, interferon-γ and interleukin-1ß was observed by monocytes at transplantation compared to healthy individuals. Remarkably, three months post-transplant, in presence of potent immunosuppressive drugs and despite improved kidney function, interferon-γ, tumor necrosis factor-α and interleukin-10 production capacity still remained significantly increased. CONCLUSION: Our data demonstrate a skewed balance towards pro-inflammatory CD16+ monocytes that is present at the time of transplantation and retained for at least 6 months after transplantation. This shift could be one of the important drivers of early post-transplant cellular immunity.

Hard corona composition and cellular toxicities of the graphene sheets.

Graphene nanomaterials are recognized as one of the most promising nanomaterials because of their unique and highly attractive physicochemical properties (e.g., thermal conductivity, superlative mechanical strength, and ultrahigh surface-to-volume ratios). It is well established that when nanomaterials interact with biological medium, biomolecules and in particular proteins attach to their surfaces, which form a complex between surface of nanoparticles and proteins called corona. Thus, the interaction of the biological system with the nanomaterials depends on the composition of the protein layer, rather than the surface characteristics of the nanomaterials itself. Although there is a significant increase of interest in the application of graphene in medical science, there has been a little attention to the nanotoxicological aspects of these newly developed materials. For this reason, we aimed to investigate whether the effect of the interactions between graphene-sheets with various human plasma concentrations (i.e. both in vitro (cells/tissues) and in vivo simulating states) is toxic. The results showed that by increasing the human plasma concentration, the affinity of proteins with low molecular weights to graphene-sheets surface is significantly increased. Fluorescence microscopy of Hela and Panc-1 cell lines showed a reduction of nuclei number and an increase of reactive oxygen species (ROS) production respectively after a longer incubation of graphene-sheets with plasma proteins. ROS production was higher in Panc-1 cell line, when used as protein source for graphene-sheets than HeLa cell line.

Significance of cell "observer" and protein source in nanobiosciences.

It is well understood that when nanoparticles (NPs) enter a biological medium, their surface is coated by various proteins; thus, the interaction of the living systems with the NPs depends on the composition of the protein layer, rather than the surface characteristics of the nanoparticle itself. However, there are several neglected parameters in protein-NP interactions (e.g., the key role of the protein source) that should be addressed. The composition of the protein corona is recognized as having a crucial influence on the delivery of NPs into cells, which is important in therapeutic applications and in nanotoxicology; however, the effect of "cell observer" (cell type) is poorly understood. This study probed the effects of different protein sources (fetal bovine serum [FBS] and human plasma [HP]) on the composition and protein thickness of the hard corona formed at the surface of superparamagnetic nanoparticles (SPIONs) with various sizes and surface chemistries. The results show that the hard corona can change quite considerably as one passes from the biophysicochemical properties of nanoparticles and protein sources (e.g., FBS and HP) appropriate to in vitro cell/tissue studies to those appropriate for in vivo studies. These changes in the hard corona have deep implications for in vitro-in vivo extrapolations. In addition, we probed the "cell observer" effect on the uptake and toxicity of SPIONs with the same protein corona composition to highlight the effect of cell type in nanobiosciences. The particles interacted with various cell lines. We find that without consideration of the "cell observer" effect, the cellular targeting/toxicity of NPs is inherently imprecise; thus, a deep understanding of both the protein corona composition and the "cell observer" effect offer a way to predict NP dosage for therapy and for the study of nanotoxins.

Characterization of coagulation factor synthesis in nine human primary cell types.

The coagulation/fibrinolysis system is essential for wound healing after vascular injury. According to the standard paradigm, the synthesis of most coagulation factors is restricted to liver, platelets and endothelium. We challenged this interpretation by measuring coagulation factors in nine human primary cell types. FX mRNA was expressed by fibroblasts, visceral preadipocytes/adipocytes and hepatocytes, but not in macrophages or other cells. All cells expressed FVIII except endothelial cells. Fibroblasts, endothelial cells and macrophages produced thrombomodulin but not FV. Interestingly, vascular-related cells (platelets/monocytes) that expressed FV did not express FX and vice versa. Monocytes expressed FV, FVIII and FXIIIA, which are positive regulators of clot formation, but these cells also contained thrombomodulin, a negative regulator of coagulation. Our data show that the expression of coagulation factors is much more complex than previously thought, and we speculate that this intricate regulation of coagulation factor expression is necessary for correct fine-tuning of fibrinogenesis versus fibrinolysis.

Hedgehog signalling as an antagonist of ageing and its associated diseases.

Hedgehog is an important morphogenic signal that directs pattern formation during embryogenesis, but its activity also remains present through adult life. It is now becoming increasingly clear that during the reproductive phase of life and beyond it continues to direct cell renewal (which is essential to combat the chronic environmental stress to which the body is constantly exposed) and counteracts vascular, osteolytic and sometimes oncological insults to the body. Conversely, down-regulation of hedgehog signalling is associated with ageing-related diseases such as type 2 diabetes, neurodegeneration, atherosclerosis and osteoporosis. Hence, in this essay we argue that hedgehog signalling is not only important at the start of life, but also constitutes an important anti-geriatric influence, and that enhanced understanding of its properties may contribute to developing rational strategies for healthy ageing and prevention of ageing-related diseases.

Propionic acid affects immune status and metabolism in adipose tissue from overweight subjects.

BACKGROUND: Adipose tissue is a primary site of obesity-induced inflammation, which is emerging as an important contributor to obesity-related diseases such as type 2 diabetes. Dietary fibre consumption appears to be protective. Short-chain fatty acids, e.g. propionic acid, are the principal products of the colonic fermentation of dietary fibre and may have beneficial effects on adipose tissue inflammation. MATERIALS AND METHODS: Human omental adipose tissue explants were obtained from overweight (mean BMI 28·8) gynaecological patients who underwent surgery. Explants were incubated for 24 h with propionic acid. Human THP-1 monocytic cells were differentiated to macrophages and incubated with LPS in the presence and absence of propionic acid. Cytokine and chemokine production were determined by multiplex-ELISA, and mRNA expression of metabolic and macrophages genes was determined by RT-PCR. RESULTS: Treatment of adipose tissue explants with propionic acid results in a significant down-regulation of several inflammatory cytokines and chemokines such as TNF-α and CCL5. In addition, expression of lipoprotein lipase and GLUT4, associated with lipogenesis and glucose uptake, respectively, increased. Similar effects on cytokine and chemokine production by macrophages were observed. CONCLUSION: We show that propionic acid, normally produced in the colon, may have a direct beneficial effect on visceral adipose tissue, reducing obesity-associated inflammation and increasing lipogenesis and glucose uptake. Effects on adipose tissue as a whole are at least partially explained by effects on macrophages but likely also adipocytes are involved. This suggests that, in vivo, propionic acid and dietary fibres may have potential in preventing obesity-related inflammation and associated diseases.

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-ß 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.

Regulation of adipokine production in human adipose tissue by propionic acid.

BACKGROUND: Dietary fibre (DF) has been shown to be protective for the development of obesity, insulin resistance and type 2 diabetes. Short-chain fatty acids, produced by colonic fermentation of DF might mediate this beneficial effect. Adipose tissue plays a key role in the regulation of energy homeostasis, therefore, we investigated the influence of the short-chain fatty acid propionic acid (PA) on leptin, adiponectin and resistin production by human omental (OAT) and subcutaneous adipose tissue (SAT). As PA has been shown to be a ligand for G-protein coupled receptor (GPCR) 41 and 43, we investigated the role of GPCR's in PA signalling. MATERIALS AND METHODS: Human OAT and SAT explants were obtained from gynaecological patients who underwent surgery. Explants were incubated for 24 h with PA. Adipokine secretion and mRNA expression were determined using ELISA and RT-PCR respectively. RESULTS: We found that PA significantly stimulated leptin mRNA expression and secretion by OAT and SAT, whereas it had no effect on adiponectin. Furthermore, PA reduced resistin mRNA expression. Leptin induction, but not resistin reduction, was abolished by inhibition of Gi/o-coupled GPCR signalling. Moreover, GPCR41 and GPCR43 mRNA levels were considerably higher in SAT than in OAT. CONCLUSIONS: We demonstrate that PA stimulates expression of the anorexigenic hormone leptin and reduces the pro-inflammatory factor resistin in human adipose tissue depots. This suggests that PA is involved in regulation of human energy metabolism and inflammation and in this way may influence the development of obesity and type 2 diabetes.

Increased plasma macrophage inflammatory protein (MIP)-1alpha and MIP-1beta levels in type 1 Gaucher disease.

Pancytopenia, hepatosplenomegaly and skeletal complications are hallmarks of Gaucher disease. Monitoring of the outcome of therapy on skeletal status of Gaucher patients is problematic since currently available imaging techniques are expensive and not widely accessible. The availability of a blood test that relates to skeletal manifestations would be very valuable. We here report that macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, both implicated in skeletal complications in multiple myeloma (MM), are significantly elevated in plasma of Gaucher patients. Plasma MIP-1alpha of patients (median 78 pg/ml, range 21-550 pg/ml, n=48) is elevated (normal median 9 pg/ml, range 0-208 pg/ml, n=39). Plasma MIP-1beta of patients (median 201 pg/ml, range 59-647 pg/ml, n=49) is even more pronouncedly increased (normal median 17 pg/ml, range 1-41 pg/ml, n=39; one outlier: 122 pg/ml). The increase in plasma MIP-1beta levels of Gaucher patients is associated with skeletal disease. The plasma levels of both chemokines decrease upon effective therapy. Lack of reduction of plasma MIP-1beta below 85 pg/ml during 5 years of therapy was observed in patients with ongoing skeletal disease. In conclusion, MIP-1alpha and MIP-1beta are elevated in plasma of Gaucher patients and remaining high levels of MIP-1beta during therapy seem associated with ongoing skeletal disease.

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 3beta-hydroxysteroid content, which we hypothesized to mediate the Smo inhibition. Indeed, the inhibition of 3beta-hydroxysteroid synthesis impaired Ptch1 action on Smo, whereas adding the 3beta-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 3beta-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.