Adipocytokines mark insulin sensitivity in euthyroid ... - Springer Link

Acta Diabetol (2013) 50:73–80 DOI 10.1007/s00592-012-0399-9

ORIGINAL ARTICLE

Adipocytokines mark insulin sensitivity in euthyroid Hashimoto’s patients Anna Solini • Angela Dardano • Eleonora Santini Antonio Polini • Fabio Monzani

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Received: 2 February 2012 / Accepted: 30 April 2012 / Published online: 26 May 2012 Ó Springer-Verlag 2012

Abstract The relationship between inflammation, Hashimoto’s thyroiditis (HT) and insulin resistance is still controversial. In this regard, a pretty complete evaluation of adipocytokines levels in patients with HT has not been performed so far. We assessed retinol binding protein-4 (RBP4), adipocyte-fatty acid binding protein (A-FABP), neutrophil gelatinase–associated lipocalin (NGAL) and tumor necrosis factor-a (TNFa) levels in 93 euthyroid HT patients and 51 healthy controls (CTL), also evaluating the possible correlation between adipocytokines levels and markers of insulin resistance. No significant differences between HT patients and CTL in fasting plasma glucose and insulin levels, and HOMA index were observed. HT patients had significantly higher RBP4, NGAL and A-FABP levels than CTL, while TNFa levels did not differ between the two groups. In HT patients, RBP4 was significantly related with fT3 and fT4 levels, while A-FABP with fT4 only. Moreover, in HT patients, either RBP4 or A-FABP was directly associated with plasma insulin and HOMA index. Circulating levels of these adipocytokines were not influenced by the presence of antithyroid peroxidase or antithyroglobulin autoantibodies or only one of them, neither by autoantibodies titer. In conclusion, euthyroid HT patients are characterized by a peculiar inflammatory response of the adipose tissue, apparently related to an early reduction in insulin sensitivity and to serum thyroid hormone levels, although within the normal range. These results suggest that HT patients with high RBP4 and A-FABP levels might deserve a particular

A. Solini (&) A. Dardano E. Santini A. Polini F. Monzani Department of Internal Medicine, University of Pisa, Via Roma, 67, 56100 Pisa, Italy e-mail: [email protected]

attention, being potentially more exposed to develop insulin resistance and increased cardiovascular risk. Keywords Adipocitokines Hashimoto thyroiditis Insulin sensitivity

Introduction Retinol binding protein-4 (RBP4), a 21-kDa protein synthesized in the liver and adipose tissue, has an important role in modulating systemic insulin action and metabolic homeostasis in insulin-resistant states [1, 2]. Elevated RBP4 levels have been described in several conditions characterized, at least in part, by a certain degree of insulin resistance, like type 2 diabetes, hypertension and polycystic ovary syndrome [3–5]. More recently, it has been suggested that other adipocytokines pertaining to the same class of RBP4 might mediate metabolic abnormalities of these clinical conditions; among these, neutrophil gelatinase–associated lipocalin (NGAL) and adipocyte-fatty acid binding protein (A-FABP) have garnered attention. The former, a small 25-kDa stress protein released from injured tubular cells after various damaging stimuli, is recognized as one of the most promising biomarkers of incoming kidney injury [6, 7]. Moreover, in patients with chronic kidney disease (CKD), NGAL closely reflects the entity of renal impairment and represents a strong and independent risk marker for progression of CKD [8]. NGAL is highly expressed by agents that promote insulin resistance while is reduced by thiazolidinediones [9], and its gene expression is increased in obese individuals [1, 10]. Increased serum NGAL levels, independently associated with coronary heart disease (CHD), have been recently described as useful marker for assessing CHD risk [11].

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A-FABP, important intracellular carrier of free fatty acids (FFA) and modulator of their bioavailability, is a determinant of insulin sensitivity in obese women [12] and predicts either the development of diabetes [13] or the metabolic syndrome in adults and adolescents [14, 15]. Several direct and indirect evidence support the relationship between insulin resistance and thyroid function [16–19]; despite that, defects in insulin secretion or action have been not fully elucidated in Hashimoto’s thyroiditis, one of the most common thyroid diseases, where a certain degree of insulin resistance has been only sporadically described [20, 21], sometimes due to the coexistence of polycystic ovary syndrome [22]. Tumor necrosis factor-a (TNFa), a pro-inflammatory cytokine involved in the pathogenesis of insulin resistance, has been implicated in the cytotoxic mechanisms in autoimmune thyroid diseases [23]. In this regard, thyroid tissues obtained from Hashimoto’s disease patients show higher mRNA levels of TNFa as compared to controls [24]; however, plasma TNFa levels are inconsistently increased in these patients [25, 26]. Some other adipocyte-secreted proteins regulating energetic metabolism have been measured in children with autoimmune thyroiditis [27], but a pretty complete evaluation of these adipocytokines in adult patients with autoimmune thyroid dysfunction has not been performed so far, despite the relationship between inflammation, autoimmune thyroiditis and insulin resistance is still controversial. Therefore, aims of our study were to assess a complete panel of adipocytokines in euthyroid subjects affected by Hashimoto’s thyroiditis and healthy controls and to evaluate their possible correlation with insulin sensitivity in these individuals.

Subjects and methods Subjects One hundred and sixteen euthyroid subjects with neodiagnosed Hashimoto’s thyroiditis (HT), with positive antithyroid peroxidase antibody (TPOAb) and/or antithyroglobulin antibody (TgAb) titer, were consecutively recruited among those attending the outpatient thyroid clinic of the Department of Internal Medicine in Pisa Medical School. Inclusion criteria were age\70 years, absence of known diabetes (fasting plasma glucose lower than 126 mg/dl and absence of therapy) and any concomitant acute or chronic inflammatory disease. Obese (BMI [ 30 kg/m2) subjects, smokers and patients with hypertension or dyslipidemia were excluded from the study. As controls (CTL), 51 euthyroid healthy volunteers matched for age, gender, blood pressure and BMI were recruited. The study protocol was approved

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Acta Diabetol (2013) 50:73–80

by the Ethics Committee of the University of Pisa, and all participants gave their written informed consent. On the day of the study, subjects attended our clinical research center after an overnight (10–12 h) fast to undergo a complete clinical examination. Height and weight were recorded, and blood pressure values were measured in the sitting position with a mercury sphygmomanometer after a 10-min rest. Blood samples were collected from the antecubital vein, and serum and plasma aliquots were obtained. Serum was analyzed for glucose, total cholesterol, triglycerides, HDL cholesterol, creatinine and thyroid profile; plasma was kept at -70 °C for late analysis of cytokines. In order to select only euthyroid patients, those showing TSH or fT3 values below or above the normal range were excluded from further analysis; therefore, 93 patients were finally considered. Analytical techniques Fasting plasma glucose was measured by the hexokinase method. The levels of total cholesterol and triglycerides were determined by an enzymatic colorimetric assay, with the use of an RA-1000 analyzer (Technicon Instruments Ltd, Terrytown, NY, USA). HDL cholesterol was measured enzymatically from the supernatant after precipitation of other lipoproteins with dextran sulfate magnesium. Levels of LDL cholesterol were calculated by the Friedewald formula. A modification of the Jaffe´ method was used to determine serum creatinine. Plasma insulin was assayed by a specific radioimmunoassay (RIA) (Linco Research, St Charles, MO, USA). The homeostasis model assessment for insulin resistance (HOMA index) was computed using the formula: fasting plasma glucose concentration (millimoles per liter) 9 fasting serum insulin level (milliunits per liter)/22.5. Serum fT3 and fT4 levels were measured by RIA, and TSH by ultrasensitive immunoradiometric assay (IRMA, Techno-Genetics, Milan, Italy). TgAb were measured by IRMA (Biocode, Sclessin, Belgium), and TPOAb by RIA (Sorin Biomedica, Saluggia, Italy). Normal ranges were as follows: fT4, 8.6–18.6 pg/ml; fT3, 2.1–4.6 pg/ml; TSH, 0.3–3.6 lU/ml; TgAb, less than 50 IU/ml; TPOAb, less than 10 IU/ml. Plasma RBP4 levels were determined by a commercially available ELISA (R&D System GmbH, Wiesbaden-Nordenstadt, Germany) with an intra-assay coefficient of variation (CV) of 4.4 % and an inter-assay CV of 5.0 %. We validated the results by Western blot analysis in a subset of randomly selected controls and patients with thyroiditis (ten in each group). Briefly, sera were diluted 1:20 in SDSPAGE buffer and heated at 100 °C for 5 min. Samples (5 ll) and molecular weight markers were electrophoresed on 15 % Trys-Glycine SDS-PAGE gels and transferred to


PVDF membrane (Millipore, Billerica, MA, USA). After a blocking step using BSA 3 % in TTBS (TBS and Tween20 0.05 %) for 1 h at room temperature, blots were washed three times in TTBS and incubated overnight at 4 °C with primary antibody anti-human RBP4 (ab57620 Abcam, Cambridge, MA, USA) diluted 1:400. The bands detection was performed incubating the blot with horseradish-peroxidase-conjugated secondary antibody (AP308P Chemicon, Temecula, CA, USA) diluted 1:4,000 for 1 h at room temperature, followed by enzymatic chemiluminescence kit (34075 Pierce Biotechnology, Rockford, IL, USA). The obtained agreement between plasma RBP4 quantities measured by ELISA and Western blotting was satisfying (r = 0.68, p \ 0.005). NGAL levels were determined using a human Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA), with a 2.2 % intra-assay and a 7.5 % inter-assay variation coefficient. A-FABP levels were measured by an ELISA kit (Biovendor Laboratory Medicine, Modrice, Czech Republic) with an intra-assay variation coefficient of 1.5 %. TNFa levels were measured using an ELISA kit (R&D Systems, Minneapolis, USA), with an intra-assay and inter-assay variation coefficient of 8.7 and 5.0 %, respectively. All assays were run in duplicate. Statistics Data are expressed as mean ± SD or median and IQR as appropriate. Differences between groups were tested using the Mann–Whitney U test or the Student’s t test. Categorical variables were compared by the v2 test. Spearman correlation analysis was used to examine the association between adipocytokines concentration and other clinical and biochemical parameters. Multiple linear regression analysis using adipocytokines as dependent variables was conducted to determine the relative contributions made by each variable to the outcome variable; age, BMI, systolic blood pressure (SBP), insulin and HOMA index were used as independent variables. Significant independent variables were chosen using the stepwise variable selection method. A p value of \0.05 was accepted to indicate statistical significance. Data were analyzed using SPSS for Windows 10.0 (SPSS Inc., Chicago, IL).

Results Clinical and metabolic characteristics of the two study groups are reported in Table 1. There were no significant differences between HT patients and CTL in fasting plasma glucose and indices of insulin resistance (fasting insulin level, HOMA index). Regarding lipid profile, no difference was observed between the two groups, with the exception

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Table 1 Clinical characteristics and thyroid profile of controls (CTL) and patients with Hashimoto’s thyroiditis (HT) CTL (n = 51)

HT (n = 93)

p

Age (years)

48.1 ± 6.2

46.3 ± 14.6

ns

Gender (% M/F)

(16/84)

(14/86)

ns

BMI (kg/m2)

25.7 ± 1.3

25.3 ± 4.9

ns

Systolic pressure (mmHg)

132 ± 10

128 ± 17

ns

Diastolic pressure (mmHg) Fasting glucose (mg/dl)

78 ± 9 85 ± 9

80 ± 10 89 ± 16

ns ns

Serum insulin (lU/ml)

8.5 ± 4.4

7.4 ± 6.4

ns

HOMA index

1.78 ± 0.90

1.65 ± 1.55

ns

Total cholesterol (mg/dl)

206 ± 18

194 ± 42

ns

HDL cholesterol (mg/dl)

56 ± 7

59 ± 12

ns

LDL cholesterol (mg/dl)

124 ± 22

116 ± 37

ns

Triglycerides (mg/dl)

120 ± 25

101 ± 36

0.0007

Creatinine (mg/dl)

0.88 ± 0.14

0.83 ± 0.16

ns

fT3 (pg/ml)

2.89 ± 0.98

2.92 ± 0.55

ns

fT4 (pg/ml)

12.55 ± 3.60

11.90 ± 2.56

ns

TSH (lU/ml)

1.49 (1.14)

1.60 (1.71)

ns

TgAb (yes/no)

0/51

64/29

–

TPOAb (yes/no)

0/51

74/19

–

Data are expressed as mean ± SD or median (IQR)

of serum triglycerides, significantly higher in CTL, even within the normal range in both groups. Circulating levels of the four adipocytokines, together with a representative WB of RBP4 protein expression in the two groups of subjects, are shown in Fig. 1. HT patients had significantly higher RBP4, NGAL and A-FABP levels with respect to CTL, while TNFa levels did not differ between the two groups. Among HT patients, adipocytokine levels did not differ in relation to the presence of TPOAb or TgAb, or both (Table 2); no relationship was found between Ab titer and adipocytokine concentrations. In HT patients, but not in CTL, RBP4 level was significantly related with fT3 and fT4 value, while A-FABP with fT4 only (Fig. 2). No relationship was observed between thyroid hormones and both TNFa and NGAL levels, either in CTL or HT patients. To better clarify the relationship between thyroid function and autoimmunity in modulating circulating adipocytokine levels, we have re-analyzed data adding to the CTL group 25 patients carrying nonautoimmune thyroid dysfunction (9 with subclinical hypothyroidism and 16 with subclinical hyperthyroidism) matched for clinical characteristics and to the euthyroid HT patients 20 Hashimoto patients with subclinical hypothyroidism or hyperthyroidism. The relationships between RBP4 and fT3 and fT4 were still not significant (p = 0.73 and p = 0.76) in the former group, while in autoimmune subjects, the relationships were confirmed as statistically significant (adjusted R2 0.032, p = 0.033 for fT3 and

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Fig. 1 Circulating levels of RBP4, NGAL, A-FABP and TNFa in Hashimoto’s patients (HT) and controls (CTL). Representative WB analysis showing RBP4 protein expression in plasma of two CTL and two HT patients is also reported. Data are mean ± SD. *p Between 0.001 and 0.0001 versus CTL

adjusted R2 0.057, p = 0.006); the same was for the relationship between A-FABP and fT4 (adjusted R2 0.086, p = 0.014). Interestingly, in HT patients but not in CTL, either RBP4 or A-FABP was directly associated with plasma insulin levels and HOMA index (Fig. 3). When we divided HT patients on the basis of their median RBP4 level (above and below 15.2 lg/ml), beside the expected higher fT3, fT4 and insulin levels, those above the cutoff showed significantly higher BMI (26.3 ± 5.4 vs. 24.4 ± 4.2 kg/m2, p = 0.05) and diastolic blood pressure values (81.7 ± 9.1 vs. 77.4 ± 10.1 mmHg, p = 0.03) than those below the cutoff. A direct correlation between A-FABP and systolic blood pressure (adjusted R2 0.066, p = 0.0077) was also observed. No relationships emerged for TNFa or NGAL and the examined clinical variables in HT patients and CTL. In multiple regression analysis, after adjustment for age, BMI and systolic blood pressure, RBP4 remained significantly related to insulin levels (adjusted R2 0.222, p = 0.0093) and HOMA index (adjusted R2 0.214, p = 0.0112); the same was true for A-FABP (adjusted R2 0.272, p = 0.0031 for insulin and adjusted R2 0.258, p = 0.0229 for HOMA, respectively). Discussion Several studies have evaluated the link between thyroid function and insulin sensitivity, with conflicting results. In

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short-term hypothyroidism, no relevant alterations of insulin sensitivity, assessed by HOMA or Matsuda index, have been described [28, 29], while in another study, directly estimating the degree of insulin sensitivity by insulin clamp, a marked reduction in insulin sensitivity has been reported in either subclinical or overt hypothyroidism [21]. We show here for the first time that euthyroid subjects with Hashimoto’s thyroiditis and normal glucose tolerance are characterized by increased levels of some adipocytokines strictly related to their degree of insulin sensitivity. In HT patients, but not in controls, a strict direct relationship was found between some of these biomarkers (RBP4 and A-FABP) and either fasting insulin levels or HOMA index, a common clinical proxy of insulin resistance. Since Hashimoto’s patients were not overweight, obesity or body fat per se cannot explain the metabolic abnormalities observed in these individuals, also because either fasting insulin levels or HOMA index was within the normal range, and not significantly different from controls. Increased RBP4 levels have been previously reported in subclinical hypothyroidism [30]; however, the authors described a relationship with TSH that we could not confirm, in face of a strict relationship with fT3 and fT4 levels. This might be due to the fact that we selected only euthyroid patients, with serum hormone values similar to those of controls. However, adding to euthyroid HT, a small group of patients with autoimmune subclinical thyroid dysfunction, we were able to confirm the relationship


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Table 2 Plasma adipocytokine levels in HT patients with the presence (pos) or absence (neg) of antithyroid peroxidase autoantibodies (TPOAb) or antithyroglobulin autoantibodies (TgAb), or presence of both

RBP4 (lg/ml)

TPOAb pos

TPOAb neg

p

TgAb pos

TgAb neg

p

Both Ab pos

One Ab pos

p

16.3 ± 9.3

19.3 ± 12.9

ns

17.8 ± 10.7

15.0 ± 8.4

ns

17.1 ± 9.8

16.7 ± 10.5

ns

NGAL (ng/ml)

41.2 ± 8.3

41.6 ± 7.2

ns

41.6 ± 8.4

40.6 ± 7.3

ns

41.6 ± 8.9

41.0 ± 7.2

ns

A-FABP (ng/ml)

24.4 ± 6.0

22.9 ± 7.4

ns

24.2 ± 6.6

23.7 ± 5.6

ns

24.8 ± 6.2

23.4 ± 6.3

ns

1.6 ± 0.8

1.7 ± 1.0

ns

1.6 ± 0.8

1.8 ± 0.9

ns

1.5 ± 0.7

1.7 ± 0.9

ns

TNFa (pg/ml)

Data are expressed as mean ± SD Fig. 2 Linear relationship between RBP4 and fT3 levels (a), RBP4 and fT4 levels (b) and A-FABP and fT4 levels (c) in HT patients

between adipocytokines and thyroid hormones, which persists after adjustment for age, BMI and systolic blood pressure; on the other hand, this relationship was not found in subjects without autoimmunity, irrespective of the presence of subclinical thyroid dysfunction. This may suggest that an early, subclinical impairment of insulin sensitivity, likely present in Hashimoto’s patients and whose RBP4 levels might be a marker, could be mainly attributable to the autoimmune disease, rather than to the degree of thyroid function. The potential role of RBP4 as early marker of insulin resistance in these patients is further reinforced by the fact that BMI and blood pressure values, even within the normal range, were significantly higher in patients with RBP4 levels above the median value. A novel finding deserving attention is the increased A-FABP levels found in Hashimoto’s patients, at concentrations that were even higher than those observed in subjects with high cardiovascular risk, like patients with angiographycally documented coronary disease [31]. In

this view, A-FABP may reflect a potentially increased CV risk in Hashimoto’s patients, independently from the traditional risk factors. However, we do not have any measurable parameter on the status of the coronary or carotid district in these patients, beside the fact that none of them had a previous personal history of angina or myocardial infarction or was assuming any medication for CHD. On the other hand, the positive relationship observed between A-FABP and fT4 is difficult to explain, given that the link between this adipocytokine and thyroid function has never been explored in humans. A thyroid hormone effect on hepatic FABP content has been demonstrated in rats, where hyperthyroidism is coupled with FABP increment and hypothyroidism with its reduction [32]. Overall, these data confirm a pathophysiological link between thyroid status, and this transport protein that, obviously, needs to be further investigated. Beside its role as possible marker of insulin resistance, NGAL has been recently recognized as a protein possibly involved in the process of tumor development; in the

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Fig. 3 Linear relationship of RBP4 and A-FABP concentrations with insulin (a–c) and HOMA index (b–d) in HT patients

thyroid, it may influence survival of thyroid neoplastic cells by controlling their iron uptake [33]. In patients with hypo or hyperthyroidism, to normalize thyroid function does not affect NGAL levels [34], but this is, to our knowledge, the first report in euthyroid Hashimoto’s patients. The abovereported link between NGAL and cancer development suggests caution in interpreting this very preliminary result, and further studies should better address this issue in larger groups of subjects by adequate follow-up. The lack of difference in TNFa levels, a cytokine largely produced by adipose tissue and strictly related to insulin resistance, might be due to the fact that the two groups were perfectly matched for BMI. Another explanation could be that, differently from what reported in thyroid dysfunction, where increased TNFa levels have been described [25], our patients had a normal thyroid function, condition likely exerting a beneficial effect on the subclinical inflammatory state that usually accompanies autoimmune diseases. More specifically, Hashimoto’s thyroiditis is considered a welldefined clinical entity, but it is widely assumed that hypothyroidism of various degree is the main, if not the only cause of the clinical symptoms or signs; Erden et al. propose it as a systemic disorder, characterized by a low-grade systemic inflammation [35], and in this setting, recent data provide new insights into its clinical features, suggesting a novel role for autoimmunity per se [36, 37]. Thyroid autoantibodies against thyroid peroxidase and thyroglobulin are specifically present in patients with

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Hashimoto’s thyroiditis. In vitro, TPO antibodies induce release of pro-inflammatory cytokines from phagocytic cells [38]; however, no previous studies have compared adipocytokine levels in Hashimoto’s patients according to the type of autoantibody. We show here that adipokine levels are similar in patients with the presence of either TgAb or TPOAb, or both; moreover, the Ab titer does not correlate with adipocytokine levels, thus suggesting that the disease per se might influence this inflammatory response of the adipose tissue. In conclusion, HT patients are characterized by a peculiar inflammatory response of the adipose tissue, related to an early reduction in insulin sensitivity and to serum thyroid hormone levels, although within the normal range. These results, when confirmed in larger cohorts of patients, might have a future clinical relevance: in fact, HT patients with high RBP4 and A-FABP levels might deserve a particular attention, being potentially more exposed to develop insulin resistance and increased cardiovascular risk.

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