Endocrine Research 681

Interacting Effects of TSH and Insulin on Human Differentiated Adipocytes

Authors

D. Felske1, 2, A. Gagnon1, 2, A. Sorisky1, 2

Affiliations

1

Key words ▶ Akt ● ▶ protein kinase C ● ▶ lipolysis ●

Abstract

 Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada  Departments of Medicine and of Biochemistry, Microbiology & Immunology, University of Ottawa, Ontario, Canada



Subclinical hypothyroidism, characterized by an isolated rise in TSH serum levels with normal thyroid function, is a pro-inflammatory state associated with insulin resistance. Adipocytes express TSH receptors, but it is not known if TSH can directly inhibit insulin signaling. Using primary human differentiated adipocytes, we examined the effects of TSH on insulin-stimulated Akt phosphorylation, and whether conventional PKC (cPKC) were involved. The effect of insulin on TSH-stimulated lipolysis was also investigated.

Introduction

▼ received 24.09.2014 accepted 20.11.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1395673 Published online: December 12, 2014 Horm Metab Res 2015; 47: 681–685 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Dr. A. Sorisky Ottawa Hospital Research Institute 501 Smyth Road Ottawa Ontario K1H 8L6 Canada Tel.:  + 1/613/737 8899 Fax:  + 1/613/737 8803 [email protected]

Adipocytes express TSH (thyroid stimulating hormone) receptors, and are increasingly recognized as extra-thyroidal targets of TSH [1, 2]. Human adipocytes stimulated by TSH in culture undergo lipolysis, and release interleukin (IL) 6 and monocyte chemoattractant protein-1 [3–6]. When TSH is acutely administered to patients on thyroid hormone treatment who have been treated for thyroid cancer (thyroidectomy and radioablation) for cancer surveillance, the rise in TSH levels induces a brief induction of metabolic and vascular stress. Increases in serum levels of IL-6, tumor necrosis factor (TNF) α, lipoperoxide, leptin, and FFA occur, as well as a decrease in endothelium-dependent relaxation [5, 7–9]. Chronic isolated rises in TSH levels are seen with subclinical hypothyroidism, characterized by a compensatory elevation in TSH levels that allow a mildly failing thyroid gland to preserve normal thyroid hormone levels. Despite maintenance of thyroid function in these patients, they have an elevated risk for cardiovascular disease, with elevations in C-reactive protein, IL-6, and free fatty acids (FFAs) [10–12]. Nevertheless, clinical

TSH inhibited insulin-stimulated Akt phosphorylation in adipocytes by 54 %. TSH activated cPKC, and Gö6976, a PKCα and -β1 inhibitor, prevented the inhibitory effect of TSH on the insulin response. Insulin reduced the ability of TSH to activate cPKC and to stimulate lipolysis. Our data reveal novel interactions between TSH and insulin. TSH inhibits insulin-stimulated Akt signaling in a cPKC-dependent fashion, whereas insulin blocks TSH-stimulated cPKC activity and lipolysis. TSH and insulin act on differentiated human adipocytes to modulate their respective intracellular signals.

uncertainty persists regarding when, or even if, to treat subclinical hypothyroidism [13], so studies to understand how TSH influences adipocyte responses may inform future recommendations on treatment. In contrast, overt hypothyroidism (thyroid hormone levels below normal despite elevated TSH levels), which can disturb adipocyte metabolism and induce insulin resistance in complex ways, [14], is routinely treated with thyroid hormone replacement. Insulin resistance has also been observed in patients with subclinical hypothyroidism in some studies [15, 16], although not all [17], perhaps due to differences in the populations studied. TSH might indirectly lead to insulin resistance through chronic elevations in IL-6 and/or FFAs, observed in subclinical hypothyroidism, and recognized inducers of insulin resistance [11, 12]. However, a direct and acute role for TSH on insulin signaling in adipocytes has not yet been investigated. Our objective was to determine whether TSH directly inhibits insulin signaling in human adipocytes. Conversely, we examined the effects of insulin on TSH-stimulated lipolysis.

Felske D et al. TSH and Insulin Action in Human Adipocytes …  Horm Metab Res 2015; 47: 681–685

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682 Endocrine Research



Human subcutaneous preadipocyte isolation and culture

Human abdominal subcutaneous samples were obtained from 13 consenting patients (1 male; 13 females) undergoing elective abdominal surgery (approved by the Ottawa Health Science Network Research Ethics Board). The mean age of the patients was 53 ± 9 years and the mean body mass index was 33 ± 10 ( ± SD). The stromal preadipocytes were isolated as previously described [18]. Connective tissue and capillaries were removed by dissection and tissue was digested with collagenase CLS type 1 (200 U/g of tissue; Worthington), followed by sequential size filtration through 200, 100, 50, and 25 μm filters. Stromal cells were exposed to red cell lysis buffer (155 mM NH4Cl, 5.7 mM K2HPO4, 0.1 mM EDTA, pH 7.3) to remove contaminating erythrocytes. Preadipocytes were cultured in Dulbecco modified Eagle medium (DMEM; Hyclone) supplemented with 10 % FBS and antibiotics [0.1 mg/ml streptomycin (Life Technologies), 100 U/ ml penicillin (Life Technologies), and 50 U/ml nystatin (Calbiochem)]. Preadipocytes were expanded in growth medium for a maximum of 3 passages before differentiation or cryopreserved until needed.

Human subcutaneous adipocyte differentiation and acute stimulation

Preadipocytes were seeded at a density of 3 × 104 cell/cm2 and differentiation was induced using growth medium supplemented with 0.85 μmol/l insulin (Sigma), 100 μmol/l indomethacin (Sigma), 0.5 μmol/l dexamethasone (Steraloids), and 0.25  mmol/l isobutylmethylxanthine (Sigma) for 14 days. Approximately 60–70 % of the cells differentiated into adipocytes. The differentiation medium was removed and the cells were placed in growth medium for 2 days prior to treatment. Adipocytes were stimulated for 0–30 min with 100 nmol/l insulin or vehicle control (0.01 N HCl) in DMEM supplemented with 4 % fatty acid free BSA (Roche), 0.1 mg/ml streptomycin, and 100 U/ml penicillin. TSH (bovine; 5 mU/ml; Sigma) was added either together or 1 h prior to insulin treatment. Cells were lysed in Laemmli buffer [19] containing 5 mmol/l sodium pyrophosphate, 50 mmol/l sodium fluoride, 5 mmol/l EGTA, and 1 mmol/l sodium orthovanadate. Where indicated, adipocytes were pretreated with 1 μmol/l Gö6976 (Calbiochem; PKCα and -β1 inhibitor), or vehicle (0.1 % DMSO) for 1 h prior to treatment with TSH. Protein was quantified by the modified Lowry method (BioRad), using BSA as standard.

Lipolysis

Differentiated adipocytes were stimulated for 4 h with vehicle control (water), 5 mU/ml bovine TSH, in the presence or absence of 100 nM insulin in phenol-red free DMEM supplemented with 4 % fatty acid-free BSA, 0.1 mg/ml streptomycin, and 100 U/ml penicillin. Media were collected for glycerol measurements, and cells were lysed in Laemmli buffer for protein determination, as described above. Glycerol was quantified by colorimetric assay using Glycerol Reagent A (Sigma).

Statistical analysis

Data were analyzed using two-way ANOVA followed by Tukey’s post-hoc tests. p-Values 

Interacting Effects of TSH and Insulin on Human Differentiated Adipocytes.

Subclinical hypothyroidism, characterized by an isolated rise in TSH serum levels with normal thyroid function, is a pro-inflammatory state associated...
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