AnneMarie Gagnon

Macrophage‐conditioned medium inhibits the activation of cyclin‐dependent kinase 2 by adipogenic inducers in 3T3‐L1 preadipocytes

Macrophage infiltration into adipose tissue, associated with obesity, is thought to contribute to abnormal adipose tissue remodeling, low‐grade inflammation, and insulin resistance. Medium conditioned by macrophages (MacCM) inhibits 3T3‐L1 and human adipocyte differentiation, as well as early adipogenic cell cycle events including MCE and retinoblastoma protein (Rb) phosphorylation. Our objective was to determine if the inhibition of Rb phosphorylation was linked to changes in cell cycle‐related proteins. We treated 3T3‐L1 preadipocytes with adipogenic inducers for 24u2009h in control medium versus J774A.1‐MacCM. The differentiation‐induced mRNA and protein expression of cyclin A, an activator of cyclin‐dependent kinase (cdk) 2 which phosphorylates Rb, was inhibited by 82% and 73%, respectively, by J774A.1‐MacCM; adipogenic expression of Myc, a transcriptional regulator of cyclin A, was also suppressed significantly. Consistent with the reduction in cyclin A levels, the activation of cdk2 by adipogenic inducers was inhibited by 75% by J774A.1‐MacCM. J774A.1‐MacCM also lowered levels of cyclins D1 and D2. Inhibition studies demonstrated that platelet‐derived growth factor, an anti‐adipogenic factor found in J774A.1‐MacCM, was not responsible for the inhibitory effect on differentiation. The anti‐adipogenic effect of J774A.1‐MacCM was resistant to proteinase K and heat treatment, and was present in a <3u2009kDa fraction. Our data indicate that J774A.1‐MacCM interferes with the upregulation of cyclin A levels and cdk2 activity that are required for Rb phosphorylation and MCE in 3T3‐L1 adipogenesis. J. Cell. Physiol. 226: 2297–2306, 2011.

TSH signaling pathways that regulate MCP-1 in human differentiated adipocytes

OBJECTIVEnAdipose tissue is an extra-thyroidal thyroid-stimulating hormone (TSH) target. Increases in lipolysis and in expression and release of interleukin-6 (IL-6) occur in TSH-stimulated adipocytes, and levels of circulating free fatty acids and IL-6 rise following TSH administration to patients with previous thyroidectomy and radioablation for thyroid cancer. Our first objective was to compare how TSH stimulates protein kinase A (PKA) and inhibitor of κB (IκB) kinase (IKK)-β. Our second objective was to investigate whether TSH induces other cytokines besides IL-6.nnnMETHODSnTSH stimulation of either CHO cells expressing human TSH receptor or human abdominal subcutaneous differentiated adipocytes.nnnRESULTSnSignaling studies showed TSH increased NADPH oxidase activity, and either diphenyleneiodonium (oxidase inhibitor) or N-acetyl cysteine (scavenger of reactive oxygen species) reduced IKKβ phosphorylation. Phosphorylation of protein kinase C-δ, an upstream regulator of NADPH oxidase, was increased by TSH, and rottlerin (PKCδ inhibitor) reduced TSH-stimulated IKKβ phosphorylation. TSH upregulated monocyte chemoattractant protein-1 (MCP-1) mRNA expression and the release of MCP-1 protein in human abdominal differentiated adipocytes. H89 (PKA inhibitor) and sc-514 (IKKβ inhibitor) each blocked TSH-stimulated MCP-1 mRNA expression and protein release, suggesting PKA and IKKβ participate in this pathway.nnnCONCLUSIONSnThese data provide new information about TSH signaling in human differentiated adipocytes, and add to the evidence that TSH is a pro-inflammatory stimulus of adipocytes.

IKKβ and the anti-adipogenic effect of platelet-derived growth factor in human abdominal subcutaneous preadipocytes

To clarify how anti-adipogenic factors act on preadipocytes to inhibit their differentiation, we compared preadipocyte signaling responses generated by platelet-derived growth factor (PDGF; anti-adipogenic) versus insulin (pro-adipogenic). PDGF, but not insulin, stimulated the phosphorylation of inhibitor of kappaB kinase beta (IKKbeta) in a time-dependent manner. This PDGF-dependent phosphorylation event was inhibited by 60% (P<0.05) when the cells were pretreated with wortmannin, indicating a requirement for the phosphatidylinositol (PI) 3-kinase/AKT pathway. IKKbeta phosphorylation by PDGF was neither accompanied by IkappaBalpha degradation nor NF-kappaB activation. PDGF inhibited human adipocyte differentiation, assessed by triacylglycerol accumulation (75% reduction; P<0.01) and by fatty acid synthase protein expression (60% reduction; P<0.05); these responses were no longer apparent in the presence of sc-514, a selective inhibitor of IKKbeta. Our data describe a novel PDGF response in human preadipocytes that involves the pro-inflammatory kinase IKKbeta and demonstrate that it is required for the inhibition of adipogenesis.

Macrophage-induced preadipocyte survival depends on signaling through Akt, ERK1/2, and reactive oxygen species

Obesity is associated with adipose tissue remodeling, characterized by macrophage accumulation, adipocyte hypertrophy, and apoptosis. We previously reported that macrophage-conditioned medium (MacCM) protects preadipocytes from apoptosis, due to serum withdrawal, in a platelet-derived growth factor (PDGF)-dependent manner. We have now investigated the role of intracellular signaling pathways, activated in response to MacCM versus PDGF, in promoting preadipocyte survival. Exposure of 3T3-L1 preadipocytes to J774A.1-MacCM or PDGF strongly stimulated Akt and ERK1/2 phosphorylation from initially undetectable levels. Inhibition of the upstream regulators of Akt or ERK1/2, i.e. phosphoinositide 3-kinase (PI3K; using wortmannin or LY294002) or MEK1/2 (using UO126 or PD98509), abrogated the respective phosphorylation responses, and significantly impaired pro-survival activity. J774A.1-MacCM increased reactive oxygen species (ROS) levels by 3.4-fold, and diphenyleneiodonium (DPI) or N-acetyl cysteine (NAC) significantly inhibited pro-survival signaling and preadipocyte survival in response to J774A.1-MacCM. Serum withdrawal itself also increased ROS levels (2.1-fold), and the associated cell death was attenuated by DPI or NAC. In summary, J774A.1-MacCM-dependent 3T3-L1 preadipocyte survival requires the Akt and ERK1/2 signaling pathways. Furthermore, ROS generation by J774A.1-MacCM is required for Akt and ERK1/2 signaling to promote 3T3-L1 preadipocyte survival. These data suggest potential mechanisms by which macrophages may alter preadipocyte fate.

mTORC1 activates SREBP-2 by suppressing cholesterol trafficking to lysosomes in mammalian cells

Significance Through unknown mechanisms mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi to produce mature SREBP-2, which translocates to the nucleus to act as a transcription factor. Low ER cholesterol is a well-known inducer of SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing ER cholesterol levels. We report that, in cultured mammalian cells, an increase in mTORC1 activity is accompanied by a decrease in ER cholesterol and by SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with higher ER cholesterol and lower SERBP-2 activity. We demonstrate that, by suppressing autophagy and by maintaining endosomal recycling, mTORC1 actively prevents membrane-derived cholesterol from reaching lysosomes, thereby reducing cholesterol ER and activating SREBP-2. mTORC1 is known to activate sterol regulatory element-binding proteins (SREBPs) including SREBP-2, a master regulator of cholesterol synthesis. Through incompletely understood mechanisms, activated mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi where SREBP-2 is cleaved to translocate to the nucleus and activate gene expression for cholesterol synthesis. Low ER cholesterol is a well-established trigger for SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing cholesterol delivery to the ER. We report here that mTORC1 activation is accompanied by low ER cholesterol and an increase of SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with a rise in ER cholesterol and a decrease in SERBP-2 activity. This rise in ER cholesterol is of lysosomal origin: blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from increasing and, consequently, SREBP-2 is activated without mTORC1 activation. Furthermore, when mTORC1 activity is low, cholesterol is delivered to lysosomes through two membrane trafficking pathways: autophagy and rerouting of endosomes to lysosomes. Indeed, with dual blockade of both pathways by Atg5−/− and dominant-negative rab5, ER cholesterol fails to increase when mTORC1 activity is low, and SREBP-2 is activated. Conversely, overexpressing constitutively active Atg7, which forces autophagy and raises ER cholesterol even when mTORC1 activity is high, suppresses SREBP-2 activation. We conclude that mTORC1 actively suppresses autophagy and maintains endosomal recycling, thereby preventing endosomes and autophagosomes from reaching lysosomes. This results in a reduction of cholesterol in the ER and activation of SREBP-2.

Thyroid-stimulating hormone acutely increases levels of circulating pro-coagulant microparticles.

Thyroid-stimulating hormone (TSH) acts on extra-thyroidal targets. When recombinant human (rh)TSH is administered to patients treated for thyroid cancer (thyroidectomy and radioablation) to screen for recurrence, metabolic and vascular stress occurs. This is indicated by elevations in levels of interleukin (IL)-6, C-reactive protein (CRP), oxidative stress and free fatty acids (FFA), as well as a decrease in endothelium-dependent relaxation. Microparticles (0 1–1 0 lm; or microvesicles) form by outward plasma membrane blebbing followed by shedding from cells. They display pro-coagulant activity by presenting surface phosphatidylserine. Platelet microparticles predominate; other sources include erythrocytes, leucocytes and endothelial cells. Elevations in total, and endothelial, microparticles correlate with indices of inflammation and vascular injury and may independently predict cardiovascular disease. Our objective was to determine whether rhTSH alters microparticle levels and procoagulant activity. Patients (12 women, six men) with a mean ( SD) age of 52 12 years and body mass index (BMI) of 29 5 kg/m, treated by total thyroidectomy and radioablation (14 papillary carcinoma, four follicular carcinoma), and on L-thyroxine therapy, were recruited (Ottawa Health Sciences Network Research Ethics Board, #2006558). Patients were disease free, that is no thyroglobulin response to rhTSH. They received two intramuscular doses of rhTSH (0 9 mg) on days 1 and 2 without discontinuation of L-thyroxine therapy. Blood was drawn on the mornings of days 1 (baseline; before rhTSH), 3 and 5 (serum separator tubes; BD Biosciences, Mississauga, ON, Canada). Samples were centrifuged at 1300 g for 10 min, and supernatants (sera) were used to measure TSH and free thyroxine (reference ranges 0 3–5 6 mU/l and 7–17 pmol/l, respectively) by AutoIA (Abbot DxI) in The Ottawa Hospital. Sera for microparticle and pro-coagulant measurements were frozen; they were thawed once for aliquoting and refrozen at 80 °C until thawed for analysis. For microparticle analysis, thawed samples (150 ll) were centrifuged at 2500 g (15 min, 4 °C). Supernatants were centrifuged at 2500 g (15 min, 4 °C). Supernatants were removed, then centrifuged at 20 000 g (20 min, 4 °C) to pellet the microparticles. Supernatants were aspirated, and microparticle-containing pellets were resuspended in Annexin V-binding buffer (10 mmol/l HEPES, pH 7 4, 140 mmol/l NaCl, 2 5 mmol/l CaCl2). Samples were labelled with 0 5 lg/ml Alexa-647 conjugated Annexin V (Biolegend, San Diego, CA, USA) and FITC-labelled CD144 antibody (1:50 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min for optimal labelling. As negative controls, a subpopulation of microparticles was resuspended in Annexin Vbinding buffer lacking calcium, and a subpopulation of microparticles was labelled with FITC-conjugated IgG isotype controls. Microparticles were assessed using a MoFlo Fluorescence Activated Cell Sorter (Dako Canada Inc, Burlington, ON, Canada) as particles of <1 0 and >0 1 lm that exhibited more fluorescence than negative controls. Results were expressed as number of annexinV total microparticles/ll plasma (interassay CV 9 5%) or of annexinV/CD144 endothelial microparticles/ll (interassay CV 11 4%). Phosphatidylserine-positive microparticles were assessed using a Zymuphen MP-activity kit (Aniara). The assay utilizes immobilized annexin V to capture phosphatidylserine (PS)-expressing microparticles. Microparticles are detected by addition of coagulation factor Va, factor Xa, Ca2 + and prothrombin. The rate of thrombin production, as indicated by a chromogenic substrate (405 nm), is proportional to PS availability and thereby, microparticle concentration. Results are expressed in nmol/l of PS equivalents (interassay CV 10%). Differences between means were assessed by ANOVA with Student Newman Keul post hoc tests. Linear regression and Pearson’s correlation were used to compare rhTSH responses where indicated. P < 0 05 was considered significant. Following rhTSH injection, TSH levels (mean SD) increased from 0 5 0 9 to 98 3 7 1 mU/l (day 3) and then decreased to 17 9 4 3 mU/l (day 5). Free thyroxine values (mean SD) were 17 9 4 3 pmol/l. The level of total microparticles was 36 651 microparticles/ll at baseline, increased 1 6-fold to 57 890 microparticles/ll on day 3 and significantly increased 1 9-fold to 70 969 microparticles/ll on day 5 (Fig. 1a). Baseline pro-coagulant activity was 5 5 nmol/l PS equivalent and increased significantly by 1 5-fold to 8 5 nmol/l PS equivalent and by 1 9-fold to 10 1 nmol/l PS equivalent on days 3 and 5, respectively (Fig. 1b). Endothelial microparticles measured 7535 microparticles/ll at baseline (20 6% of total) and did not change in response to rhTSH (Fig. 1c). Increases in total microparticles and pro-coagulant activity (responses at day 5 for each person) were correlated (r = 0 70; P < 0 05). There was a negative correlation of total microparticle response at day 5 with age (r = 0 56; P < 0 05); a similar trend with pro-coagulant activity (r = 0 40, P = 0 10) did not reach significance. There was no correlation with BMI. There were too few men to compare responses by gender. As levels of total microparticles rose with rhTSH, but endothelial particles did not, the response may be due to platelet microparticles, the main subpopulation, although other microparticle populations (e.g. leucocyte) cannot be excluded. rhTSH increases platelet reactivity (soluble P-selectin and soluble CD40 ligand) and endothelial activation (soluble intercellular adhesion molecule-1, soluble E-selectin). These rhTSH-induced

Effect of High Glucose Concentration on Human Preadipocytes and Their Response to Macrophage-Conditioned Medium

OBJECTIVESnAdipose tissue expands via differentiation of preadipocytes into adipocytes (adipogenesis) and/or hypertrophy of existing adipocytes. A low adipogenic capacity promotes adipocyte hypertrophy, causing inflammatory macrophage accumulation and insulin resistance. Macrophage-conditioned medium (MacCM) inhibits adipogenesis and promotes adipocyte inflammation, but it is unknown whether these effects are altered by high glucose (HG) versus normal glucose (NG) concentrations. Our aim was to compare the effect of HG-MacCM versus NG-MacCM on human adipogenesis and adipocyte inflammation.nnnMETHODSnHuman monocyte-derived macrophages (MDMs) were placed in 5u2009mmol/L (NG) or 25u2009mmol/L (HG) glucose for 24 hours. MacCM was collected and its effect on differentiation of human subcutaneous abdominal preadipocytes and adipocyte inflammation was evaluated.nnnRESULTSnHG-MacCM, but not NG-MacCM, inhibited triacylglycerol (TG) accumulation and protein expression of peroxisome proliferator-activated receptor γ (PPARgamma) during human adipogenesis. Preadipocytes differentiated in HG-MacCM displayed a more pro-inflammatory phenotype, as assessed by increased interleukin-6 and monocyte chemotactic protein-1 (MCP-1), as well as reduced adiponectin mRNA expression. In MDMs, HG increased phosphorylation of inhibitor of kappaB kinase (IKK)-beta and decreased protein expression of inhibitor of kappaB alpha. HG also reduced protein expression of PPARgamma in MDMs. However, no MDM changes in mRNA expression of MCP-1, interleukin-1beta or tumor necrosis factor-alpha were detected. The stimulatory effect of HG-MacCM on MCP-1 expression in adipocytes was partially inhibited when MDMs were treated with sc-514 (IKKbeta inhibitor).nnnCONCLUSIONSnHigh glucose concentration accentuates the anti-adipogenic and pro-inflammatory effects of MacCM on human preadipocytes.

Thyroid-Stimulating Hormone-Stimulated Human Adipocytes Express Thymic Stromal Lymphopoietin

When recombinant human (rh) thyroid-stimulating hormone (TSH) is administered to thyroid cancer survivors, an acute extra-thyroidal effect raises pro-inflammatory cytokines and activates platelets. Thymic stromal lymphopoietin (TSLP) is a cytokine recently implicated in platelet activation. Our aim was to measure platelet microparticle levels after rhTSH stimulation in vivo, and to investigate TSLP expression in TSH-stimulated human adipocytes in culture. Blood samples for total and platelet microparticle analysis were obtained from thyroid cancer survivors before (day 1) and after rhTSH administration (day 5). Adipocytes, differentiated from stromal preadipocytes isolated from adipose tissue from surgical patients, were stimulated with TSH. TSLP mRNA expression, protein expression, and protein release into the adipocyte medium were measured. The level of platelet microparticles in thyroid cancer patients rose 5-fold after rhTSH stimulation. TSH upregulated TSLP mRNA expression in adipocytes in culture through a pathway that was inhibited by 66% by H89, a protein kinase A inhibitor. TSLP protein expression rose in response to TSH, and TSH-stimulated TSLP release into the medium was completely blocked by dexamethasone. In conclusion, TSLP is a novel TSH-responsive adipokine. Future studies will be needed to address the potential role of adipocyte-derived TSLP and whether it is linked to TSH-dependent platelet activation.