doi: 10.1161/01.ATV.0000102520.84030.A1
© 2003 American Heart Association, Inc.
Letters to the Editor |
TSH Stimulates IL-6 Secretion from Adipocytes in Culture
Department of Medicine and of Biochemistry, Microbiology & Immunology, University of Ottawa, Ottawa Health Research Institute, Ottawa, Canada
To the Editor:
Adipocytes secrete interleukin-6 (IL-6), a pro-atherogenic cytokine.1–3 However, regulation of adipocyte IL-6 secretion remains largely unknown. Adipocytes express thyroid-stimulating hormone (TSH) receptors.4–6 Because subclinical hypothyroidism, characterized by an elevated TSH level, is an independent cardiovascular disease (CVD) risk factor,7 we hypothesized that TSH stimulates adipocyte IL-6 secretion.
3T3-F442A (Dr. Howard Green, Harvard University, Boston, Mass)8 and 3T3-L1 (ATCC) cells were grown in DMEM with 10% calf serum in 35-mm dishes. Confluent 3T3-F442A preadipocytes were differentiated in control medium (DMEM with 10% fetal bovine serum [FBS]) with 1 µM insulin for 8 days.9 Confluent 3T3-L1 preadipocytes were differentiated with control medium with 1 µmol/L insulin over 8 days, and 0.25 µmol/L dexamethasone and 0.5 mmol/L isobutylmethylxanthine (IBMX) present for the first 2 days.9 Subcutaneous abdominal adipose tissue was obtained from 9 patients (4 male, 5 female) undergoing elective abdominal surgery (Ottawa Hospital Research Ethics Committee approval). Human preadipocytes were isolated by collagenase digestion and serial filtrations. Cells were seeded in 24-well plates at 3x104 cells/cm2 in DMEM with 20% FBS, grown to confluence, and then placed in serum-free differentiation medium.10 Differentiation medium was DMEM:Ham’s F12 (1:1), 33 µmol/L biotin, 17 µmol/L pantothenate, 10 µg/mL transferrin, 0.2 nM triiodothyronine, 1 µmol/L insulin, and 1 µmol/L cortisol. For the first 4 days, 0.5 mmol/L IBMX, 25 nM dexamethasone, and 5 µmol/L troglitazone (Roche) were added. The medium was then replaced every 3 days for 2 weeks, and 70% differentiation was observed.
Differentiated 3T3-F442A and 3T3-L1 adipocytes were placed in DMEM, and treated with 5 µmol/L bovine TSH (Sigma), 1 µmol/L isoproterenol, or vehicle (water) for 4 hours. Human adipocytes were placed in DMEM or DMEM with 1% calf serum and/or 10 nM insulin-like growth factor 1 (IGF-1), and treated with 0.01–5 µM bovine TSH, 1 µM highly purified bovine TSH (TSH-NIH; AFP8755B, National Institutes of Diabetes and Digestive Kidney Diseases; National Hormone and Peptide Program, Torrance, CA), 1 µM recombinant human TSH (rhTSH; Thyrogen), or vehicle (water) for 4 hours. IL-6 in the medium was measured by enzyme immunometric assay (Assay Designs, Inc.). Data were analyzed by t test or ANOVA with Tukey’s post hoc test, using GraphPad InStat version 3.00 (Graph Pad Software), with P<0.05 considered significant.
We assessed the effect of 5 µmol/L TSH on IL-6 secretion in three adipocyte models under serum-free conditions. Figure 1A shows that TSH (5 µmol/L) treatment of 3T3-F442A adipocytes for 4 hours increased IL-6 secretion from 6.5±0.8 to 31.1±2.6 (pg/mL per µg protein; mean±SEM), a 5-fold rise. Isoproterenol (1 µmol/L), which elevates IL-6 secretion in these cells,2 induced a 6.3-fold increase in IL-6 secretion, up to 41.0±6.1(pg/mL per µg protein; mean±SEM). In Figure 1B, a similar response to TSH was observed with 3T3-L1 adipocytes, in which IL-6 secretion increased by 2-fold, from 3.2±0.5 to 6.3±1.1 (pg/mL per µg protein; mean±SEM). Figure 1C demonstrates that TSH stimulated IL-6 secretion in human differentiated abdominal adipocytes by 3.5-fold, from 204±32 to 690±39 (pg/mL; mean±SEM). Although we use the term "secretion," we cannot formally rule out the possibility of a TSH-mediated inhibition of IL-6 degradation in the medium.
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To ensure specificity, we added TSH-NIH, a highly purified bovine TSH, or rhTSH to human differentiated adipocytes (Figure 2A). Control IL-6 secretion (pg/mL; mean±SEM) was 186±14. There was a 2.1±0.2-fold increase (mean±SEM) in secretion with commercial 1 µmol/L TSH. Stimulation of human adipocytes with 1 µmol/L TSH-NIH induced a 1.8±0.2-fold increase in IL-6 secretion (mean±SEM). rhTSH (1 µmol/L) increased IL-6 secretion by 1.8±0.1-fold (mean±range). TSH acts in concert with serum and IGF-1 in thyrocyte models,11 so we tested a lower concentration of TSH in this context (Figure 2B). In the presence of 1% calf serum and 10 nM IGF-1, 0.01 µmol/L TSH significantly induced adipocyte IL-6 secretion. This TSH concentration, approximately 1 mU/mL, is entirely consistent with TSH concentrations used for studies on cultured thyrocytes, an unequivocal TSH target cell.12–14 Normal circulating TSH concentrations are much lower, in the range of 1 mU/L, rising to 10 mU/L on average with subclinical hypothyroidism. Two-dimensional cell culture conditions therefore incompletely recapitulate the in vivo context for cellular targets of TSH.
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rhTSH was tested in 2 separate patients in duplicate. B, Cells were stimulated with 0.01 µM TSH for 4 hours in the presence of 1% calf serum with or without 10 nM IGF-1. Data are expressed as pg/mL (means±SEM) of 3 separate patient studies, each performed in duplicate. *P<0.05, versus control or IGF-1 alone.Subclinical hypothyroidism is characterized by a compensatory rise in TSH levels to maintain thyroid hormone levels despite a mildly failing thyroid gland. The Whickham Survey on this condition found a weak association with ECG changes; subsequent analysis did not show a link to future cardiovascular disease events, possibly due to more awareness to treat subclinical hypothyroidism.15,16 However, TSH was identified as an independent risk factor for CVD in the population-based cross-sectional Rotterdam study of more than 1000 women, with an attributable risk for myocardial infarction similar to that of other major CVD risk factors.7 This effect was independent of known CVD risk factors, including body mass index, blood pressure, and lipoproteins. The increase in IL-6 release from adipocytes by TSH reported here suggests a novel molecular mechanism by which subclinical hypothyroidism may predispose to CVD.
Acknowledgments
Acknowledgments
This work was funded by a grant-in-aid from the Heart and Stroke Foundation of Canada. AS is a Career Investigator of the Heart and Stroke Foundation of Ontario. AB holds a Doctoral Research Award from the Heart and Stroke Foundation of Canada/Canadian Institutes of Health Research. AG is supported by a Premier’s Research Excellence Award held by AS.
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