Anne Landry

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.

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.

The anti‐adipogenic effect of peripheral blood mononuclear cells is absent with PCSK9 loss‐of‐function variants

To determine the effect of (1) an oral fat load and (2) pro‐protein convertase subtilisin/kexin type (PCSK) 9 loss‐of‐function (LOF) variant status on the ability of peripheral blood mononuclear cells (PBMC) to inhibit human adipogenesis.

Dechlorane Plus increases adipogenesis in 3T3-L1 and human primary preadipocytes independent of peroxisome proliferator-activated receptor γ transcriptional activity

Background/objectivesPolybrominated diphenyl ethers (PBDEs) are chemicals that were added to consumer products to reduce flammability but were deemed toxic and bioaccumulative and were phased out of commerce. Flame retardants (FRs) such as Dechlorane Plus (DP) were introduced as replacements. DP is being produced in high volumes and is detected in the environment, human milk, and human serum. Although human exposure to DP is evident, little is known about its potential effects on human health. We and others have shown that some FRs are potential obesogens, i.e., promote adipogenesis. However, the effects of DP on adipogenesis are not known.MethodsMurine 3T3-L1 and human primary subcutaneous (Sc) and omental (Om) preadipocytes were differentiated in the presence of DP (0.001–10u2009µM) and adipogenic effects were measured. Further, the ability of DP to activate the adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) was also assessed.ResultsWe show that treatment of murine preadipocytes with DP significantly (pu2009<u20090.05) increased lipid accumulation (2.5-fold) and the mRNA expression of adipogenic markers: fatty acid binding protein 4 (Fabp4), lipoprotein lipase (Lpl), perilipin (Plin), adipsin, and adiponectin. DP also significantly (pu2009<u20090.05) increased the protein levels of selected mature adipocyte markers. We further show using luciferase reporter assays that DP increased PPARγ transcriptional activity by threefold (pu2009<u20090.05). When the PPARγ agonist was replaced by DP in the human preadipocyte differentiation cocktail, DP significantly (pu2009<u20090.05) increased the mRNA levels of adipogenic markers, PPARγ, FABP4, and PLIN in human Sc as well as Om cultures. Finally, PPARγ antagonist studies revealed that DP-mediated upregulation of adipogenic markers Fabp4 and Lpl did not occur via PPARγ activation.ConclusionThe current study shows that DP can induce adipogenesis of murine and human preadipocytes. We show that, although DP can directly activate PPARγ, its adipogenic effects may be mediated via other pathways.

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.

Elevated ChREBPβ and TXNIP Levels in Human Adipocytes Differentiated in High Glucose Concentrations

OBJECTIVESnObesity and type 2 diabetes often coexist. The effect of hyperglycemia on adipose tissue is, therefore, of interest. Although studies have shown that high glucose (HG) concentrations do not inhibit adipocyte differentiation, the resulting adipocyte phenotype has not been investigated. In particular, the levels of the glucose-responsive transcription factor carbohydrate-responsive response element binding protein (ChREBP) isoforms have not been assessed.nnnMETHODSnHuman preadipocytes were differentiated into adipocytes in either normal glucose (NG) or HG conditions. RNA and protein analyses were used to measure the expression of ChREBP isoforms, thioredoxin interacting protein (TXNIP) and lipogenic genes. Insulin-stimulated glucose uptake was measured.nnnRESULTSnHG- vs. NG-differentiated adipocytes expressed more ChREBPβ and more TXNIP at the mRNA and protein levels. There was no change in lipogenic gene expression. HG- vs. NG-differentiated adipocytes displayed an inhibition of insulin-stimulated glucose uptake.nnnCONCLUSIONSnHG-differentiated human adipocytes have distinct molecular differences and are insulin resistant. More studies are warranted to investigate potential mechanisms linking changes in ChREBPβ and TXNIP to insulin responsiveness.