Life Sciences, Vol. 30, pp. 641-650 Printed in the U.S.A.

Pergamon Press

LIPOLYTIC AND CIRCULATORY RESPONSES TO NOPADRENALINE INFUSION IN HYPOTHYROID SUBJECTS BEFORE AND DURING THYROXINE SUBSTITUTION

Urban Rosenqvist and Britta Hylander

Depts of Endocrinology and Internal Medicine, Karolinska Hospital Stockholm, Sweden

Summary

The hypothyroid state in humans is associated with a diminished lipolytic response to noradrenaline in adipose tissue in vitro. In the present study we have investigat- ed the situation in vivo in order to see if such a change in receptor response could be demonstrated in adipose tissue and in the circulatory system. The change in glycerol production rate, induced by an infusion of noradrenaline, was used as an index of adipose tissue adrenergic responsiveness. The results showed that the lipolytic response was decreased by about 50% in the hypothyroid state and that it was normalized when the substitution dose had been increased to 0.10-0.15 mg/day thyroxine. The circulatory response was monitored by measurements of blood pressure and pulse rate. The rest- ing diastolic pressure was transiently lowered by substi- tution. Similarly the rise in systolic blood pressure in- duced by 1-noradrenaline was transiently increased by substitution. Thus no clear-cut change in receptor re- sponse with substitution could be demonstrated by measur- ing BP and pulse rate only. This result could be due to the fact that the system is more complex than the adi- pose tissue.

The finding of a reduced adrenergic receptor response in vivo in the adipose tissue is in accordance and _- extends earlier findings in vitro.

In a previous series of experiments it was shown that the hypothyroid state in human subjects was accompanied by an inhibi- tion of the in vitro response to 1-noradrenaline in subcutaneous adipose tissueThe mechanism behind this change in response was found to be an enhancement of the alpha adrenergic responsive- ness which suppressed the formation of c-AMP and lipolysis (2). When the patients had become enthyroid after substitution, the adrenergic receptor response was also normalized and l-noradrena- line elicited a normal lipolytic effect. However, the patients did not consent to a study of the time course of receptor norm- alization by multiple adipose tissue biopsies. Similar effects of the hypothyroid state on the alpha adrenergic receptor responsive- ness has been observed in rabbit aorta (3) and in heart (4) using different in vitro systems. Whether such alterations in adrenergic receptor responses also exist in living hypothyroid individuals is presently not known. The clinical picture of lethargy andinability

0024-3205/82/070641-10$03.00/O Copyright (c) 1982 Pergamon Press Ltd.

642 Noradrenaline Effect in Hypothyroidism Vol. 30, No.s 7 & 8, 1982

to produce heat as well as the increased levels of plasma nor- adrenaline (5) found in the hypothyroid patient might be indi- cators of a reduced adrenergic receptor responsiveness in vivo.

The aim of the present study was to investigate if there is an altered adrenergic receptor response in hypothyroid subjects in vivo and if so how large it is and time course of recovery upon thyroxine substitution. Two organ systems containing adre- nergic receptor - adipose tissue and the circulation - were studied.

Methods

Seven hypothyroid subjects were studied before and repeatedly during substitution with thyroxine (Levaxin Nyegaard, Norway). The initial dose was 0.05 mg/day and it was increased every fourth week by 0.05 mg until the level of TSH was normal.Clinical and laboratory data on the patients are given in Table 1.

The lipolytic and circulatory responses to 1-noradrenaline was studied before substitution and after four weeks on each sub- stitution dose level. In addition the patients were studied after three months on final dose. The subjects were fasting over night and studied in the morning in the supine position. A short cath- eter was inserted into each cubital vein and kept patent by a slow infusion of saline. The patients rested for 30 min before an infusion was started containing 0.05 pg/kg/min 1-noradrenaline in saline + 0.5 mg/kg/min Vitamin C to inhibit oxidation of l- noradrenaline. The flow was maintained by a pump which delivered 60 ml/hour. The infusion was terminated after 30 min and the patient rested for another 30 min. At the start of the l-noradre- naline infusion 0.2 UCi 14C-glycerol (sp. act. 8 MCi/mmole) in 5 ml saline was rapidly injected intravenously. Plasma samples were collected in Heparinised tubes at -10, -5, and 0 min before the bolus injection of 14C-glycerol and every second min for 30 min for analysis of glycerol and 14C-glycerol. Glycerol was analysed in quintiplicates according to Chernick (6). Carbon-14 labelled glycerol was determined in 2 ml plasma according to (7) with some modification. Recovery was corrected for by addition of O.OlpCi 3H-glycerol (sp. act. 200 MCi/mmole) to the plasma aliquote. The mixture was then purified by mixing with 20 ml isopropanol and shaken for 15 min. The precipitate formed was removed by centri- fugation and the supernatant mixed with 4g Zeolite and shaken for 30 min. The mixture was then centrifuged at 1OOOxg and the super- natant mixed with 2 ml 0.25 M Ba04 and 2 ml 0.25 M ZnSO4 in order to remove proteins. After neutralization and centrifugation the supernatant was applied to an ion-exchange columne made of IRA 410. The whole eluate was collected and dried by a gentle stream of air. The residue was then disolved in 1 ml distilled water and 10 ml Aquazo?,e was added. The samples were counted in a two channel liquid scintilation counter for sufficient time give a coefficient of variation of less than 3%. for by internal standardization.

Quenching was c?irected The average recovery of C-

glycerol was 30%. Only 0.6% glucose was recovered after the puri- fication procedure when separately tested by the use of 14C- glucose.

The metabolic clearance rate (MCR), distribution volume (V) for 14C-glycerol was calculated by fhe non-compartemental method (8). The average production rate (PR) of glycerol during the in- fusion was calculated as described (9). The basal glycerol pro-

Vol. 30, No.s 7 & 8, 1982 Noradrenaline Effect in Hypothyroidism 643

duction rate was calculated by multiplying the concentration of glycerol at time 0 with the MCR obtained during the l-noradrena- line infusion.

The circulatory response to 1-noradrenaline stimulation was assessed by measurements of blood pressure and pulse rate every fifth minute during the experiment.

Analysis of Thyreotropin (TSH) and Thyroxine (T4) were made by radioimmunoassay. The normal range was: TSH t7 arb. units/ml and T4 70-150 nmol/l.

All chemicals were of highest purity and double distilled water was used throughout. The enzymes for determination of glycerol were obtained from Sigma.

Statistical comparisons were made by the use of paired student's t-test (IO).

The study was approved by the ethical- and isotope committees of the Karolinska Hospital and the subjects gave their informed consent.

Results

All subjects were hypothyroid according to both clinical and laboratory criteria (Table I). The gradual increase of the Tq dose was well tolerated by all subjects and none noted any adverse reactions during the experiment. All patients had a normal TSH level when the T4 dose was 0.15 mg/dl.

TABLE 1

Clinical and laboratory findings in the subjects tested

No. Age Y

Sex

62 m 57 f 48 m 48 m 41 f 37 f 31 f

Diagnosis* TSH T4 arb U/ml nmol/l

2 33 40 1 60 16 2 58 28 1 195 IO 2 33 29 1 14 60 1 150 IO

* I) Hashimoto's thyroiditis 2) Post 1311 treatment

The basal level of glycerolwas not significantly increased by T4 substitution although the mean values were higher (Table 2, Fig I).

The lipolytic effect of 1-noradrenaline was significantly in- creased at a T4 dose of 0.01 when the total area under the plasma glycerol curves were compared (Table 2, Fig I). When the net effect of 1-noradrenaline was calculated only the stimulation ob- served after 3 months on full substitution was significantly in- creased (Table 2).

644 Noradrenaline Effect in Hypothyroidism Vol. 30, No.s 7 & 8, 1982

Plasma glycerol p mole/ml

1

-10 -5 0 10 20 30 40 50 60

Time (mid

Tq - 0.0 3-o 0.05 H 0.10 - 0.15 - 0.15x3

FIG. 1

Effect of l-noradrenaline infused between O-30 min on plasma glycerol at different levels of Tq substitution. Mean values of

glycerol. N=7.

The MCR and basal production rate of glycerol was unchanged by substitution (Table 2). However, the lipolytic effect of l- noradrenaline as measured by the P-R was significantly stimulated by T4 substitution (Fig 2). The difference in I% of the first two points of observation and the remaining three was 0.4OkO.15 (MeanLSE) umoles/kg/min ~(0.05. Six out of the seven patients showed a full recovery between the 4th and 8th week of substitu- tion while one patient required 4 more weeks before he.recovered.

The effect of T4 substitution on blood pressure and heart rate at rest and during l-noradrenaline infilsion is shown in fig 3. Thyroxine therapy resulted in a significant lowering of the resting diastolic blood pressure at the 8th and 12th week of sub- stitution (pt0 .Ol and pXO.05). Noradrenaline increased both the systolic and diastolic blood pressure and lowered the pulse rate. When compared with the effects obtained before substitution T4 only influenced the 1-noradrenaline effect on systolic blood pressure after 12 w of treatment (pt0.05) and the diastolic pressure after 8 w (~(0.05).

Vol. 30, No.s 7 & 8, 1382

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646 Noradrenaline Effect in Hypothyroidism Vol. 30, No.s 7 & 8, 1982

Effect of NA- infusion

Net glycerol production rate vmole /min/kg

l.O-

0.8 ‘I

0.6-

0.4-

0.2 -

‘4 t I 8 12

TIME (weeks)

m/d 1 r0.15

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FIG. 2

Noradrenaline stimulated 5% of glycerol at different levels of Tq substitution. Mean+%. N=7.

Discussion

The hypothyroid state in human subjects is associated with a number of slowed down functions. The patients have difficulties to produce enough heat, maintain the blood pressure and ventilate adequately in the extreme state (11). These derangements can be- come life threatening when the patient develops myxoedema coma. Previous in vitro studies of human subcutaneous adipose tissue showed that the normal adrenergic receptor response to l-noradre- naline was absent in the hypothyroid state.

We chose to study two different organ systems which show re- sponses to l-noradrenaline stimulation in the euthyroid state. Adipose tissue contains adrenergic receptors that increases the formation of CAMP and lipolysis and alpha receptors that have an opposite action (12). Thus, the lipolytic effect of l-noradrena- line which can stimulate both types of receptors should reflect the balance between the two types of receptors. In the present study it was assumed that this dual effect of l-noradrenaline would also be present in vivo. However, since the study had to be restricted due to the limits set by the isotope committee wecould

Vol. 30, No.s 7 & 8, 1982 Noradrenaline Effect in Hypothyroidism 647

rest (-1 and during NA-infusion (0)

mmHg or

min”

160

100

80

60

40

0

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Diast BP

Pulse

+NA

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+NA

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Time (weeks)

FIG. 3

Effect of Td substitution on the circulatory response to l-noradrenaline. MeanLSE. N=7.

not use other agonists or blockers in addition to the time course studies of the blunted response to l-noradrenaline. It therefore remains to be shown whether the marked decrease in 1-noradrenaline response is in fact due to a change'in alpha-beta receptor balance as previously shown in vitro or if it only reflects a loss of beta receptors (13). In contrast to the previous in vitro results the inhibition of l-noradrenaline response observed here was not com- plete. This most probably reflects the difference in conditions between the in vivo and in vitro experimental set-up. It was most interesting to find that the normalization of the noradrenaline response occurred in all patients within a four week interval during substitution and that it was completed before or at the same time TSH became normal. This is a relatively early effect of substitution when compared with other effects of the hormone (Fig 4). The glycerol rate of production under unstimulated con- ditions as calculated from the basal concentration of glycerol and the MCR was found to be of the same range as that of previous authors (1, 7, 14).

The mechanism behind the inhibited lipolytic response in vitro to noradrenaline in adipose tissue from hypothyroid subjects

648 Noradrenaiine Effect in Hypothyroidism Vol. 30, No.s 7 & 8, 1982

THERAPEUTIC EFFECTS OF T4

therapeutic effect lipolysis TSH LDL flicker ortostatic

0

physical C, 0 fusjon reac.$on cap%city

-----------

Thyroxine dose

weeks

me/d 0.1 5

0.0

FIG. 4

Therapeutic effect of T4 substitution on different variables. Lipolysis and TSH present study, low density lipoprotein (LDL) concentration (151, flicker fusion test (16), pulse reaction in the orthostatic test (171, total work performed on an ergometer

cycle (17).

has been attributed to a change in the intracellular handling of Ca++ which in turn would influence the alpha receptor response (18, 19). It may be that the mitochondria of hypothyroid tissue in general have a reduced capacity to bind CaCf and at least in our preliminary experiments with the rat liver it took 14 days to normalize the capacity well after the tissue exhibited an'increase in oxygen consumption and the animals started to grow. The delay before restoration of adrenergic responsiveness observed in the present experiments might thus also be due to the fact that some support system for the adrenergic response must be restored be- fore the tissue can respond adequately. So far we do not know what part of the cell could serve this function in vivo.

The response of the circulatory system to T4 substitution was transient and not so distinct as that of the adipose tissue. The system is probably more complex with different feed back loops that try to overcome the effects induced by substitution and the 1-noradrenaline infusion. In order to demonstrate a specific change of the adrenergic receptor response of the circulatory

Vol. 30, No.s 7 & 8, 1982 Noradrenaline Effect in Hypothyroidism 649

system in vivo more sophisticated methods may have to be used. So far it cannot be excluded that such a change may exist since studies of the rat heart do have demonstrated an enhanced alpha receptor response and receptor number in the hypothyroid state (4).

In conclusion this study has demonstrated that the adrenergic response is blunted in vivo in adipose tissue and that it is re- stored early in the course of substitution.

Acknowledgement

This study was supported by grants from the Swedish Medical Research Council 19X-05707-02 and the Karolinska Institutet.

References

1. U. ROSENQVIST, Acta Med. Stand. suppl. 532:1-28 (1972). 2. V. GRILL, U. ROSENQVIST, Acta Med. Stand. 194:129-133 (1973). 3. U. ROSENQVIST, L-O. BOREUS, Life Science 11 part 1:595-604

(1972). 4. H.Y. CHANG, G. KLJNOS, Biochem. Biophys. Res. Comm. 100:313-320

(1981). 5. N.J. CHRISTENSEN, J. Clin. Endocrin. Metabol. 35:359-363

(1972). 6. S.S. CHERNICK. Methods in Enzymology, Vol 4:~. 627, Academic

Press, New York (1969). 7. W.N. BORTZ, J. Clin. Invest. 51:1537-1546 (1972). 8. J.H. OPPENHEIMER, H.L. SCHWARTZ, M. SURKS, J. Clin. Endocrinol.

Metab. 41:319-324 (1975). 9. E. GURPIDE. Tracer Methods in Hormone Research, p. 133,

Springer-Verlag, Berlin (1975). 10. P. ARMITAGE. Statistical methods in medical research, p. 118,

Academic Press, London .(1974). 11. J.T. NIKOLOFF, Pharmacol. Ther. Comm. 1:161-189 (1976). 12. V. GRILL, U. ROSENQVIST, Acta Med. Stand. 197:283-287 (1975). 13. M. GINSBERG, W.E. CLUTTER, S.D. SHAH, P.E. CRYER, J. Clin.

Invest. 67:1785-1791 (1981). 14. C.F. BORCHGREVINK, R.J. HAVEL, Proc. Sot. Exp. Biol. Med. 113:

946-949 (1963). 15. B. HYLANDER, U. ROSENQVIST, Acta Med. Stand. In press. 16. S. LEVANDER, U. ROSENQVIST, Neuropsychobiology 5:274-281

(1979). 17. B. HYLANDER, U. ROSENQVIST, In preparation. 18. U. ROSENQVIST, Acta Med. Stand. 196:69-73 (1974). 19. U. ROSENQVIST, Molec. Cell. Endocr. 12:111-117 (1978).

650 Noradrenaline Effect in Hypothyroidism VOL. 30, NO.S 7 & a, 1982

EDITED GENERAL DISCUSSION

Paper of Rosenqvist

Kuchel commented that Rosenqvist had reported briefly the history of a lady dying from myxedema coma in spite of treatment with thyroxine, and since primary hypothyroidism is accompanied by hypoadrenocorticism, the admin- istration of cortisol and thyroid hormones could be very helpful in such a dramatic situation. Bilezekian wondered whether thyroxine acts on the number or affinity of the a or g receptors, but Rosenqvist pointed out these deter- minations have not been done. Werner then asked if Na+ and I? metabolism had *been studied. Rosenqvist responded that thyroxine tended to increase plasma Na+ levels and decrease plasma R? levels. Resnick thought Rosenqvist's ex- periments suggest that thyroxine may act on a balance between adrenergic a or 6 receptors. He inquired what effect blockade of the a receptors might have. Rosenqvist noted that during hypothyroidism there is increased a tonus, and that also administration of phentolamine to hypothyroid patients reduces insulin release.