Effects of Thyrotropin on Peripheral Thyroid Hormone Metabolism and Serum Lipids

Abstract

Background:

Subclinical hypothyroidism is associated with dyslipidemia and atherosclerosis. Whether these effects are in part mediated via direct effects of thyrotropin (TSH) on peripheral thyroid hormone (TH) metabolism and/or concentrations of serum lipids is not clear.

Objective:

This study examined whether TSH has direct effects on peripheral TH metabolism and serum lipids.

Methods:

Eighty-two patients with differentiated thyroid cancer were retrospectively analyzed. All patients had undergone total thyroidectomy and ¹³¹I remnant ablation. During follow-up, two successive injections of recombinant human TSH (rhTSH) were administered to patients on a stable dose of levothyroxine. In all patients, TSH, thyroxine (T4), free T4 (fT4), triiodothyronine (T3), reverse T3 (rT3), total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, apolipoprotein B, lipoprotein(a), and triglyceride levels were measured immediately before the first and approximately 72 hours after the second injection of rhTSH.

Results:

After rhTSH stimulation, T3 values decreased (from 1.91 to 1.81 nmol/L; p < 0.001). T4, fT4, and rT3 did not change. After rhTSH, median apolipoprotein B increased from 0.90 to 0.92 g/L (p = 0.03), lipoprotein(a) from 0.21 to 0.24 g/L (p < 0.001), and triglycerides from 1.98 to 2.50 mmol/L (p < 0.001). Serum high-density lipoprotein cholesterol decreased from 0.98 to 0.81 mmol/L (p < 0.001). Multiple regression analysis showed that the changes in lipids were most closely associated with the decrease in T3 levels.

Conclusions:

TSH has direct effects on peripheral TH metabolism by decreasing T3 levels in levothyroxine-treated thyroidectomized patients. This decrease in T3 levels is accompanied by unfavorable changes in serum lipids.

Introduction

Subclinical hypothyroidism is defined by an elevated thyrotropin (TSH), with thyroxine (T4) and triiodothyronine (T3) levels within the normal range. This condition is associated with increased cardiovascular risk and elevated levels of total and low-density lipoprotein cholesterol (LDL-C) (1 –7). It is likely that this association is caused by the slightly lower thyroid hormone (TH) levels in patients with subclinical hypothyroidism, rather than by the elevated TSH itself (8,9). However, extrathyroidal TSH receptors have been shown in extraocular muscle, bone, fat tissue and liver, indicating that TSH may also have extrathyroidal effects (10 –12). Studies in liver cells and rats suggest that TSH directly upregulates cholesterol synthesis via increased hepatic expression of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (11,12). This led to the hypothesis that part of the effects of subclinical hypothyroidism on lipid levels may also be mediated via direct effects of TSH.

Approximately 20% of circulating T3 in healthy subjects is secreted by their thyroid gland. The major fraction is produced by deiodination of T4 in peripheral tissues (13). Intracellular T4 is converted to bioactive T3 by type 1 and type 2 deiodinase (D1, D2). Inactivation of TH is mediated mainly by type 3 deiodinase (D3), which converts T4 to reverse T3 (rT3) and T3 to 3,3′-diiodothyronine (14). Many factors have been identified that influence peripheral TH metabolism, such as starvation, medication, illness, and long-term TSH-suppressive levothyroxine (LT4) therapy (15,16). Although TSH has been shown to increase hepatic conversion of T4 to T3 in perfused rat liver (17), direct effects of TSH on peripheral TH metabolism have not been reported in humans.

Patients treated for differentiated thyroid cancer (DTC) by thyroidectomy and ¹³¹I ablation completely lack functional thyroid tissue. Consequently, all serum T3 in these patients results from conversion of T4 in peripheral tissues. In the follow-up of DTC, most patients may undergo a recombinant human TSH (rhTSH) stimulation test as part of a dynamic risk stratification. This stimulation test provides an ideal model to study the effects of TSH because of the constant T4 supply. The present study took advantage of this model to determine whether TSH has direct effects on peripheral TH metabolism and serum lipid levels.

Methods

Patients and treatment

Initial therapy consisted of total thyroidectomy and radioiodine (¹³¹I) remnant ablation. Patients were on TSH suppressive LT4 therapy aiming at a TSH level of ≤0.1 mIU/L. Success of ablation was evaluated after about 6 months by means of rhTSH stimulated thyroglobulin measurements. On day 1, baseline nonfasting blood was drawn for measurement of thyroid function tests (TFT) and lipids. Subsequently, 0.9 mg of rhTSH was administered intramuscularly on days 1 and 2. On day 5, a second nonfasting blood sample was drawn.

Data of the rhTSH tests with a negative thyroglobulin response and negative thyroglobulin antibodies (TgAb) were retrospectively analyzed. Based on these results, patients were deemed to be completely athyroid.

The present study employed serum samples collected in an ethically approved study at the Erasmus MC (MEC 2012-561). Furthermore, leftover remnant sera from regular clinical procedures from the University Hospital Wuerzburg from the 2009–2013 time frame were analyzed. The study was performed in accordance with the German regulations on the use of serum sample remnants after diagnostic use. Samples were anonymized before analysis.

TFT

TFT and lipid levels before and after rhTSH administration were analyzed in the same run. Serum TSH (reference range 0.4–4.3 mIU/L), free T4 (fT4; reference range 11–25 pmol/L), thyroglobulin, and TgAb were measured as part of routine clinical practice. In Erasmus MC, TSH, thyroglobulin, and TgAb were measured using the Immulite 2000XPi platform (Siemens, Los Angeles, CA) with functional sensitivities of 0.01 mIU/L for TSH, 0.2 μg/L for thyroglobulin, and 2.2 IU/mL for TgAb. Free T4 was measured using the Vitros ECiQ (Ortho-Clinical Diagnostics, Rochester, NY), with a functional sensitivity of 0.88 pmol/L. At the University Hospital Wuerzburg, TSH, fT4, as well as free T3 (fT3; reference range 2.7–7.6 pmol/L) were also measured using the Immulite 2000XPi platform (Siemens). The intra-assay coefficient of variation for fT3 was 3.2–7.0%. Serum thyroglobulin levels were determined using the immunoradiometric assay from BRAHMS (functional sensitivity of 0.4 μg/L) (9). TgAb levels were assessed using the direct chemoluminometric VARELISA method (Thermo Fisher Scientific, BRAHMS, Uppsala, Sweden).

In all samples, total T4 (reference range 58–128 nmol/L) and T3 levels (reference range 1.43–2.51 nmol/L) were measured using the Vitros ECiQ (Ortho-Clinical Diagnostics). Reverse T3 (reference range 0.21–0.54 nmol/L) was measured with an in-house radioimmunoassay (18) with an intra-assay coefficient of variation of 4.2–8.7%. T3/T4 × 100 (reference range 1.42–3.05), T3/rT3 (reference range 2.65–7.65), and rT3/T4 × 100 (reference range 0.15–0.44) ratios were calculated. These ratios are relatively insensitive to variations in protein binding and are considered to reflect peripheral deiodinase activities (19).

Lipids

Direct measurement of total cholesterol (reference range 2.9–6.5 mmol/l), LDL-C (reference range 2.59–4.12 mmol/L), high-density-lipoprotein cholesterol (HDL-C; reference range 0.9–1.1 mmol/L), and triglycerides (reference range <2.0 mmol/L) were measured using standard laboratory techniques. Apolipoprotein B (apoB; reference range 0.60–1.33 g/L) was measured by immunoturbidimetry on a c311 automatic analyzer (Roche Diagnostics, Basel, Switzerland). Plasma lipoprotein(a) (Lp[a]; reference range <0.30 g/L) concentrations were measured using a particle-enhanced immunoturbidimetric assay, which is largely independent of the apo(a) kringel IV type 2 repeat number (DiaSys Diagnostic System, GmbH, Holzheim, Germany) (20). LDL-C was also calculated via the Friedewald formula [total cholesterol – HDL-C – (0.45 × triglycerides)] (21) and non-HDL-C via the formula (total cholesterol – HDL-C) (22).

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics for Windows v23 (IBM Corp., Armonk, NY). Changes in TFT and lipids were analyzed using the paired-sample t-test, and if not normally distributed via the paired Wilcoxon signed-rank test. Power calculations showed that with the largest sample that was available, using an alpha of 0.05 and a power of 80%, a statistically significant effect size of 0.3 would be able to be detected (23). Normal distribution was ascertained using the Kolmogorov–Smirnov test, and residuals that were not normally distributed were log transformed. Multiple linear regression models were used to study if the association of TSH with serum lipids was (partly) mediated via changes in T3. Multiple comparisons were accounted for using the Benjamini and Hochberg false discovery procedure (24). Corrected p-values of 0.05 were considered significant.

Results

A total of 82 patients were studied: 56 patients from the University Hospital Wuerzburg and 26 patients from the Erasmus MC, Rotterdam. Thirty-two patients were male. The median age was 44 years (interquartile range [IQR] = 35–59 years). None of the patients used medication that may influence TH metabolism (e.g., corticosteroids or amiodarone).

TH

As expected, serum TSH concentrations increased strongly after rhTSH injection (Table 1). Despite a constant dose of LT4 and stable concentrations of fT4 and rT3, T3 concentrations decreased after rhTSH injections (Table 1). The decrease in T3/T4 ratio and T3/rT3 ratio was mainly due to the decrease in T3 values (Table 1). Additionally, serum fT3, which was measured in the clinical routine in the 51 patients included in Wuerzburg, also decreased (Table 1).

Table 1.

Change in Thyroid Function Tests After Recombinant Thyrotropin Stimulation

Variable	Reference value	Before rhTSH	After rhTSH	p
Parameter
TSH (mIU/L)^a	0.4–4.3	0.02 (0.01–1.00)	9.68 (7.30–14.6)	<0.001^***
fT4 (pmol/L)^b	11–25	23.6 (4.7)	23.6 (4.9)	0.41
T4 (nmol/L)^b	58–128	156 (41)	159 (43)	0.41
T3 (nmol/L)^b	1.43–2.51	1.91 (0.30)	1.81 (0.30)	<0.001^***
fT3 (pmol/L)^b	2.7–7.6	5.71 (1.34)	5.01 (1.05)	<0.001^***
rT3 (nmol/L)^b	0.21–0.54	0.53 (0.10)	0.53 (0.20)	0.90
Ratio
T3/T4 × 100^a	1.42–3.05	1.22 (1.01–1.44)	1.13 (0.91–1.47)	<0.001^***
T3/rT3^a	2.65–7.65	3.61 (2.96–4.63)	3.53 (2.75–4.34)	0.02^**
rT3/T4 × 100^a	0.15–0.44	0.33 (0.30–0.40)	0.33 (0.29–0.39)	0.90

Changes in TFT were analyzed using a paired-sample t-test and if not normally distributed via a paired Wilcoxon signed-rank test. False discovery rate correction for multiple comparisons was applied as proposed by Benjamini and Hochberg.

Median (IQR); ^b M (SD).

p ≤ 0.01; ^*** p ≤ 0.001.

fT4, free thyroxine; IQR, interquartile range; M, mean; SD, standard deviation; TFT, thyroid function test; TSH, thyrotropin; rhTSH, recombinant TSH; T3, triiodothyronine; T4, thyroxine; rT3, reverse T3.

Lipids

Median apoB, Lp(a), non-HDL-C, and triglycerides increased after rhTSH. Serum HDL-C decreased, whereas total cholesterol and LDL-C did not change (Table 2).

Table 2.

Changes in Nonfasting Serum Lipid Concentrations After rhTSH Injection

Variable	Reference value	Before rhTSH	After rhTSH	p
apoB (g/L)^a	0.60–1.33	0.90 (0.03)	0.92 (0.03)	<0.05^*
Total cholesterol (mmol/L)^a	2.9–6.5	5.09 (0.14)	5.17 (0.13)	0.17
HDL-C (mmol/L)^a	0.9–1.1	0.98 (0.05)	0.81 (0.05)	<0.001^***
LDL-C (mmol/L)^a	2.59–4.12	2.90 (0.10)	2.84 (0.10)	0.40
Lp(a) (g/L)^a	<0.30	0.21 (0.03)	0.24 (0.03)	<0.001^***
Triglycerides (mmol/L)^a	<2.0	1.98 (0.14)	2.50 (0.16)	<0.001^***
LDL-C-Friedewald (mmol/L)^a	2.59–4.12	3.24 (0.13)	3.29 (0.13)	0.40
Non-HDL-C (mmol/L)^a	2.0–5.4	4.10 (0.15)	4.36 (0.15)	<0.001^***

Analysis by general linear model for repeated measurements was used, and false discovery rate correction for multiple comparisons proposed by Benjamini and Hochberg was applied.

Friedewald formula: [total cholesterol – HDL-C – (0.45 × triglycerides)]; non-HDL-C formula: (total cholesterol – HDL-C).

M (SD).

p ≤ 0.05; ^*** p ≤ 0.001.

apoB, apolipoprotein B; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; Lp(a), lipoprotein(a).

Next, to determine if these changes in lipid levels could be attributed to direct effects of rhTSH or to its effects on serum T3 levels, serum T3 levels were added as a covariate to the multiple regression model. This showed that the changes in lipid levels were most closely associated with the changes in T3 and showed no evidence for an additional effect of rhTSH itself (Table 3).

Table 3.

T3 Adjusted Change in Nonfasting Serum Lipid Concentrations After rhTSH Stimulation

Variable	Reference value	Before rhTSH	After rhTSH	Adjusted p
apoB (g/L)^a	0.60–1.33	0.90 (0.03)	0.92 (0.03)	0.74
Total cholesterol (mmol/L)^a	2.9–6.5	5.09 (0.14)	5.17 (0.13)	0.74
HDL-C (mmol/L)^a	0.9–1.1	0.98 (0.05)	0.81 (0.05)	0.92
LDL-C (mmol/L)^a	2.59–4.12	2.90 (0.10)	2.84 (0.10)	0.92
Lp(a) (g/L)^a	<0.30	0.21 (0.03)	0.24 (0.03)	0.74
Triglycerides (mmol/L)^a	<2.0	1.98 (0.14)	2.50 (0.16)	0.92
LDL-C-Friedewald (mmol/L)^a	2.59–4.12	3.24 (0.13)	3.29 (0.13)	0.74
Non-HDL-C^a	2.0–5.4	4.10 (0.15)	4.36 (0.15)	0.74

Multiple regression analysis was performed in general linear model for repeated measurements, where changes in serum lipids were corrected for change in T3. False discovery rate correction for multiple comparisons was applied as proposed by Benjamini and Hochberg.

Friedewald formula: [total cholesterol – HDL-C – (0.45 × triglycerides)]; non-HDL-C formula: (total cholesterol – HDL-C).

M (SD).

Discussion

To the best of the authors' knowledge, this is the first study that provides evidence of a direct effect of TSH on peripheral TH metabolism in humans. The study was performed in a controlled setting during which patients were on a stable daily LT4 dosage. Administration of exogenous rhTSH led to a decrease in T3 and fT3 concentrations, and as a consequence also to decreased T3/T4 and T3/rT3 ratios. The results indicate that any TSH-related changes in lipid levels in vivo are mediated by a drop in (f)T3 concentrations when T3 is added as a covariate to the multiple regression model.

Peripheral TH metabolism

Unravelling the exact molecular mechanism causing the decreased T3 concentrations requires further in vitro and in vivo studies. Potentially, the decrease in T3 concentrations can be explained by a decreased conversion of T4 to T3 by D1 or D2, or by an increased degradation of T3 to rT3 by D3. Since D2 may be more important than D1 for peripheral T3 production in humans, whereas D1 may be more important than D2 for rT3 degradation (25), it is speculated that a decrease in D2 activity is the most likely explanation for this reduction in T3 levels in the presence of unaltered rT3 levels. In the case of decreased D1 activity, an increase in rT3 concentrations would be expected as well (19,26). An increased degradation by D3 would not only lead to lower concentrations of T3, but also to lower concentrations of T4, given the constant LT4 dose, and an increase in rT3 concentrations (via the degradation of T4).

Other explanations for the decrease in T3 concentrations upon rhTSH stimulation include an increased T3 degradation via nondeiodinative conjugation by forming T3 sulfate (27) and/or T3-glucuronide formation (28). These reactions would increase T3 solubility and biliary and urinary excretion. Also, altered T4 transport into T3 producing cells could explain the decrease in T3 concentrations, since this transport is the rate-limiting step for subsequent metabolism.

Most circulating T3 is bound to proteins such as thyroxine-binding globulin, albumin, and transthyretin. Given that not only total T3 but also fT3 concentrations decreased after rhTSH administration, it seems unlikely that the drop in serum T3 concentrations after rhTSH stimulation is caused by a change in T3 binding protein concentrations or affinity.

A temporary change in diet after rhTSH injections might theoretically result in lower T3 levels (14), since fasting is a known cause of low T3 syndrome. However, caloric restriction would decrease especially triglycerides (29). The increase in serum triglycerides that was identified and the lack of change in rT3 make this explanation highly unlikely. Furthermore, as most patients in this study underwent the procedure in an in-patient setting due to German nuclear medicine regulations, it was ascertained that patients did not fast between the two blood samples.

A previous study in 15 DTC patients did not show an effect of rhTSH administration on serum fT4 and fT3 concentrations, perhaps due to a lack of statistical power (30).

Lipids

In the current study, short-term exposure to high TSH concentrations was associated with a decrease in (f)T3 concentrations and, subsequently, a more unfavorable serum lipid profile.

Hypothyroidism-induced changes in serum lipids are well established and characterized by elevated total cholesterol (6,31), LDL-C (6,32), triglycerides (33), Lp(a) (34), apoB (34), and decreased HDL-C (35). These changes are mediated via changes in cholesterol biosynthesis (3-hydroxy-3-methylglutaryl-coenzym A), regulation of LDL-receptors (via sterol regulatory element-binding protein 2) (36), HDL-mediated reverse cholesterol transport to the liver (37), and biliary cholesterol excretion (38,39). The beneficial effect of TH on the lipid profile has led to the development of “liver-specific” thyromimetics such as Eprotirome. Eprotirome affects the T3 receptor-β isoform and results in a decrease in LDL-C, apoB, triglycerides, and Lp(a) (40). The current study identified a decrease in T3 and a very similar diametrically opposite effect in apoB, triglycerides, and Lp(a), which seems to support a diminished T3 stimulation.

LDL-C concentrations did not significantly change in this study, but non-HDL-C as well as apoB increased, indicating increased levels of triglyceride-rich lipoproteins such as very-low-density lipoprotein (VLDL) or intermediate-density lipoprotein. In line with this observation, others have shown a significant elevation of VLDL-C in patients with hypothyroidism (41).

Potentially, the nonfasting status had an impact on these results. However, the identified changes in lipids are much larger and not in line with extensive observational data as summarized in the European Society of Cardiology guidelines (42). The most important differences are that the nonfasting status affects triglycerides, with an increase of 0.3 mmol/L versus an increase of 0.5 mmol/L in the current study, and the calculated non-HDL-C decreases by 0.2 mmol/L compared to an increase of 0.3 mmol/L in the current study. Moreover, concentrations of HDL-C, apoB, and Lp(a) are not affected by fasting/nonfasting status in contrast to the current data.

In the current study, all patients lacked functional thyroid tissue due to surgery and radioiodine ablation therapy. Therefore, the isolated effects of rhTSH were able to be studied in a very homogenous group with a constant LT4 supply. However, this study also has several limitations. First of all, in contrast to the short serum half-life of triglycerides (43) and VLDL-C (44) of only a few hours, the half-life of the other lipoproteins is as long as several days (45,46). As a consequence, the steady state of serum concentration of most lipoproteins was not yet reached in this study. A maximal effect would probably be found after two weeks (47,48), but the clearance rate and the effect of two injections of rhTSH versus continuous TSH stimulation are difficult to predict. The timeframe of five days was, however, sufficient to identify significant changes in the current study. A significant change in LDL clearance can occur within one day through a change in the activities of hepatic microsomal cholesterol 7alpha-hydroxylase and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (49). Changes in Lp(a) concentrations over several days have been shown to be mainly determined by alterations in the rate of production in a kinetic study with radiolabeled Lp(a) (45).

Second, statistical modeling can only give insight into possible associations. In other experiments, a direct effect of TSH on serum lipids has been suggested, but a possible confounding effect of a decrease in T3 concentrations has not been investigated (12,50,51). Further research with direct testing is necessary to unravel fully the pathophysiologic mechanisms of TSH and TH action on lipid metabolism.

Third, the current study was performed in a specific patient population without a control group, and the data were, in part, collected retrospectively. Moreover, the possibility cannot formally be excluded that the rhTSH injections did not result, for example, in cytokine release and secondary changes in TH metabolism and action (52). Therefore, further studies in larger placebo-controlled cohorts including individuals other than thyroid cancer patients are required.

Conclusion

RhTSH administration results in a decrease of serum T3 concentrations in LT4-treated thyroidectomized patients, which in turn affect serum lipid concentrations toward a more unfavorable profile. These data suggest a direct effect of TSH on peripheral T3 production. Replication is needed, and future studies should further clarify by which molecular mechanisms TSH affects peripheral TH metabolism.

Footnotes

Author Disclosure Statement

F.A.V. is a consultant to Bayer Healthcare and Sanofi Genzyme and has received speaker honoraria from Genzyme and Diasorin. R.P.P. has received lecture and consultancy fees from Sanofi Genzyme, and lecture fees from Goodlife Fertility BV and IBSA (Institute Biochemique SA). No competing financial interests exist for the remaining authors.

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