Regulators of thymic stromal lymphopoietin production by human adipocytes.

Thymic stromal lymphopoietin (TSLP) is a cytokine that is known to play a role in inflammatory conditions, especially asthma and atopic dermatitis. It is also recognized to be expressed in human adipose tissue. TSLP production from human adipocytes is stimulated by thyroid-stimulating hormone (TSH). This study aimed to identify TSH-dependent signaling routes that regulate TSLP, to determine if TSLP production is stimulated by other cytokines (IL-1ß and TNF-α), and to examine if TSLP production depends on the adipose depot. Human abdominal differentiated adipocytes were stimulated with TSH, IL-1ß, or TNF-α. Activation of cell signaling kinases was measured by phospho-immunoblot analysis, and TSLP in medium was assessed by ELISA. TSLP responses from abdominal subcutaneous and omental adipocytes were compared. TSH-stimulated TSLP secretion from subcutaneous adipocytes was enhanced by IBMX (raises cAMP levels) and was blocked by UO126 (inhibitor of MEK1/2-ERK1/2). TSLP secretion was stimulated by IL-1ß and by TNF-α. SC-514 (inhibitor of IKKß/NF-κB) only reduced the former. There was no effect of SB203580 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) on the stimulation by TSH, IL-1ß or TNF-α. Interferon-γ inhibited TSLP responses to TSH, IL-1ß, and TNF-α; IL-4 only blocked the response to TNFα. Intra-abdominal omental adipocytes also release TSLP in response to TSH, IL-1ß, and TNF-α. We conclude TSLP is produced by human differentiated adipocytes derived from subcutaneous or omental depots in response to a variety of agonists. Further studies will be needed to understand what role it may play in adipose biology.

Elevated Carbohydrate Response Element-Binding Protein Beta (ChREBPß) and Thioredoxin Interacting Protein (TXNIP) Levels in Human Adipocytes Differentiated in High Glucose Concentrations.

OBJECTIVES: Obesity 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. METHODS: Human 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. RESULTS: HG- vs. NG-differentiated adipocytes expressed more ChREBPß 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. CONCLUSIONS: HG-differentiated human adipocytes have distinct molecular differences and are insulin resistant. More studies are warranted to investigate potential mechanisms linking changes in ChREBPß and TXNIP to insulin responsiveness.

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.

Human apolipoprotein A-II protects against diet-induced atherosclerosis in transgenic rabbits.

OBJECTIVE: Apolipoprotein (apo) A-II is the second major apo of high-density lipoproteins, yet its pathophysiological roles in the development of atherosclerosis remain unknown. We aimed to examine whether apo A-II plays any role in atherogenesis and, if so, to elucidate the mechanism involved. METHODS AND RESULTS: We compared the susceptibility of human apo A-II transgenic (Tg) rabbits to cholesterol diet-induced atherosclerosis with non-Tg littermate rabbits. Tg rabbits developed significantly less aortic and coronary atherosclerosis than their non-Tg littermates, while total plasma cholesterol levels were similar. Atherosclerotic lesions of Tg rabbits were characterized by reduced macrophages and smooth muscle cells, and apo A-II immunoreactive proteins were frequently detected in the lesions. Tg rabbits exhibited low levels of plasma C-reactive protein and blood leukocytes compared with non-Tg rabbits, and high-density lipoproteins of Tg rabbit plasma exerted stronger cholesterol efflux activity and inhibitory effects on the inflammatory cytokine expression by macrophages in vitro than high-density lipoproteins isolated from non-Tg rabbits. In addition, ß-very-low-density lipoproteins of Tg rabbits were less sensitive to copper-induced oxidation than ß-very-low-density lipoproteins of non-Tg rabbits. CONCLUSIONS: These results suggest that enrichment of apo A-II in high-density lipoprotein particles has atheroprotective effects and apo A-II may become a target for the treatment of atherosclerosis.

ABCA1 protein enhances Toll-like receptor 4 (TLR4)-stimulated interleukin-10 (IL-10) secretion through protein kinase A (PKA) activation.

BACKGROUND: ABCA1 is known to suppress proinflammatory cytokines. RESULTS: ABCA1 activates PKA and up-regulates anti-inflammatory cytokine IL-10. Elevated PKA transforms macrophages to M2-like phenotype. Disrupting lipid rafts by statins MCD, and filipin recuperates ABCA1 phenotype and likely functions downstream of ABCA1. CONCLUSION: By modulating cholesterol, ABCA1 activates PKA. This generates M2-like macrophages. SIGNIFICANCE: ABCA1 does not simply suppress inflammatory response. It promotes M2-like activation and facilitates resolution. Nonresolving inflammatory response from macrophages is a major characteristic of atherosclerosis. Macrophage ABCA1 has been previously shown to suppress the secretion of proinflammatory cytokine. In the present study, we demonstrate that ABCA1 also promotes the secretion of IL-10, an anti-inflammatory cytokine critical for inflammation resolution. ABCA1(+/+) bone marrow-derived macrophages secrete more IL-10 but less proinflammatory cytokines than ABCA1(-/-) bone marrow-derived macrophages, similar to alternatively activated (M2) macrophages. We present evidence that ABCA1 activates PKA and that this elevated PKA activity contributes to M2-like inflammatory response from ABCA1(+/+) bone marrow-derived macrophages. Furthermore, cholesterol lowering by statins, methyl-ß-cyclodextrin, or filipin also activates PKA and, consequently, transforms macrophages toward M2-like phenotype. Conversely, cholesterol enrichment suppresses PKA activity and promotes M1-like inflammatory response. As the primary function of ABCA1 is cholesterol removal, our results suggest that ABCA1 activates PKA by regulating cholesterol. Indeed, forced cholesterol enrichment in ABCA1-expressing macrophages suppresses PKA activation and elicits M1-like response. Collectively, these findings reveal a novel protective process by ABCA1-activated PKA in macrophages. They also suggest cholesterol lowering in extra-hepatic tissues by statins as an anti-inflammation strategy.

ABCA1 increases extracellular ATP to mediate cholesterol efflux to ApoA-I.

ATP-binding cassette protein A1 (ABCA1) is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apolipoprotein A-I (apoA-I). This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e., channel, pump, or flippase, remains unknown. In this study we analyzed extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of nonexpressing cells. We found that extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as wild-type ABCA1, is unable to raise extracellular ATP concentration, which suggests a casual relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e., <µM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane-bound ectonucleotidase, CD39, abolishes cholesterol efflux to apoA-I. On the basis of these results, we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

Ht31, a protein kinase A anchoring inhibitor, induces robust cholesterol efflux and reverses macrophage foam cell formation through ATP-binding cassette transporter A1.

Macrophage foam cell is the predominant cell type in atherosclerotic lesions. Removal of excess cholesterol from macrophages thus offers effective protection against atherosclerosis. Here we report that a protein kinase A (PKA)-anchoring inhibitor, st-Ht31, induces robust cholesterol/phospholipid efflux, and ATP-binding cassette transporter A1 (ABCA1) greatly facilitates this process. Remarkably, we found that st-Ht31 completely reverses foam cell formation, and this process is ABCA1-dependent. The reversal is also accompanied by the restoration of well modulated inflammatory response to LPS. There is no detectable toxicity associated with st-Ht31, even when cells export up to 20% cellular cholesterol per hour. Using FRET-based PKA biosensors in live cells, we provide evidence that st-Ht31 drives cholesterol efflux by elevating PKA activity specifically in the cytoplasm. Furthermore, ABCA1 facilitates st-Ht31 uptake. This allows st-Ht31 to effectively remove cholesterol from ABCA1-expressing cells. We speculate that de-anchoring of PKA offers a novel therapeutic strategy to remove excess cholesterol from lipid-laden lesion macrophages.

Cholesterol efflux to apoA-I in ABCA1-expressing cells is regulated by Ca2+-dependent calcineurin signaling.

ATP-binding cassette transporter A1 (ABCA1) is required for the lipidation of apolipoprotein A-I (apoA-I), although molecular mechanisms supporting this process remain poorly defined. In this study, we focused on the role of cytosolic Ca(2+) and its signaling and found that cytosolic Ca(2+) was required for cholesterol efflux to apoA-I. Removing extracellular Ca(2+) or chelating cytosolic Ca(2+) were equally inhibitory for apoA-I lipidation. We provide evidence that apoA-I induced Ca(2+) influx from the medium. We further demonstrate that calcineurin activity, the downstream target of Ca(2+) influx, was essential; inhibition of calcineurin activity by cyclosporine A or FK506 completely abolished apoA-I lipidation. Furthermore, calcineurin inhibition abolished apoA-I binding and diminished JAK2 phosphorylation, an established signaling event for cholesterol efflux to apoA-I. Finally, we demonstrate that neither Ca(2+) manipulation nor calcineurin inhibition influenced ABCA1's capacity to release microparticles or to remodel the plasma membrane. We conclude that this Ca(2+)-dependent calcineurin/JAK2 pathway is specifically responsible for apoA-I lipidation without directly modifying ABCA1 activity.

ABCA1-mediated cholesterol efflux generates microparticles in addition to HDL through processes governed by membrane rigidity.

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.