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Pyridine nucleotides in the thyroid
241
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
25564
P Y R I D I N E NUCLEOTIDES IN T H E THYROID VI. DPN KINASE ACTIVITY OF THYROID HOMOGENATE OBTAINED FROM SLICES INCUBATED W I T H AND WITHOUT THYROID STIMULATING HORMONE J A M E S B. F I E L D , S H E L D O N M. E P S T E I N * , A D R I E N N E BOYLE
K. R E M E R
AND C O N S T A N C E
The Clinical Research Unit and the Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. (U.S.A.) (Received O c t o b e r 29th, 1965)
SUMMARY
Thyroid slices incubated with and without TSH have been homogenized and assayed for DPN kinase (ATP:DPN 2'-phosphotransferase, EC 2.7.1.23) activity. Such activity was increased when homogenates from TSH incubated slices were incubated with ATP and DPN and [i-14C]-6-phosphogluconate oxidation measured as an indication of TPN formation. In similar experiments when TPN was measured directly, homogenates obtained from TSH incubated slices also had greater TPN levels than homogenates prepared from control slices. However, in these experiments since net synthesis of TPN was not observed, the results could also be explained by decreased rate of destruction as well as by increased synthesis of TPN. Subcellular fractionation of thyroid slices localized most of the DPN kinase to the nuclear fraction while the increased TPN of TSH incubated slices was in the supernatant. The TSH stimulation of glucose oxidation was not inhibited b y a concentration of actinomycin D which caused a 9 ° % reduction in [SA*C]adenine incorporation into nucleic acids. Vasopressin, which has been reported to directly stimulate thyroxine release from the thyroid, did not stimulate glucose oxidation in thyroid slices. TSH did not influence glucose oxidation of gastric mucosa and submandibular gland, two tissues which also share with thyroid the ability to concentration iodide but are not TSH responsive in terms of this parameter.
INTRODUCTION
The stimulation of glucose oxidation in thyroid slices induced by TSH has been attributed to the increased TPN observed in such slices1, 2. The increased TPN was associated with an equivalent decrease in DPN (ref. I) and could not be accounted for by changes in T P N H (ref. 3). These results suggested an effect of TSH on the enzyme DPN kinase (ATP:DPN 2'-phosphotransferase, EC 2.7.1.23) which converts * P o s t d o c t o r a l Fellow, U.S. P u b l i c H e a l t h Service.
Biochim. Biophys. Acta, 121 (1966) 241-249
242
J.B. FIELD et al.
DPN and ATP to TPN. New protein synthesis apparently was not involved since puromycin did not inhibit responsiveness to TSH although incorporation of [i-t4C!leucine into protein was reduced 98 % (ref. 2). Moderate reductions of thyroidal DPN induced by Carzinophilin did not modify TSH stimulation of glucose oxidation or TPN synthesis 4 indicating that DPN was not limiting in TPN synthesis and that TSH did not act by influencing DPN levels. The present studies were undertaken to assess the effects of TSH on the thyroid gland DPN kinase reaction in hopes of further elucidating the mechanism of action of TSH. EXPERIMENTAL PROCEDURE
The method of preparation and incubation of thyroid slices has been described 5. Thegastric mucosae were separated from the rest of the stomach by blunt dissection and sliced with a Stadie-Riggs microtome. The effect of prior incubation of thyroid slices with TSH on the subsequent activity of DPN kinase in homogenates was studied as follows. Approx. 9oo mg of thyroid slices were incubated in IO ml of Krebs-Ringer bicarbonate buffer with and without TSH (o.5 units/ml) for 45 min. Following incubation a Io % homogenate in glass-distilled water was prepared, i ml of each homogenate was extracted immediately in 3 ml of o.i M HC1 to determine the TPN concentration at the end of the slice incubation. I ml of the homogenate obtained from the control or TSH incubated slices was incubated for 9 ° min at 37 in 2.3 ml of buffer containing 50/~moles of Tris (pH 7.5), 5o/,moles nicotinamide, 5/~moles of magnesium chloride, and in appropriate tubes, o.I 5/,mole of DPN and o.16/~mole ATP. Controls without homogenate but with DPN and ATP were included in each experiment. At the end of the incubation, the reaction mixture was extracted with 2 ml of o.I M HC1 at IOO° for 3o see for TPN measurement. The effects of incubation of thyroid slices with TSH on DPN kinase activity of homogenates was also assessed by an indirect measurement of TPN based on the oxidation of [I-i4(; 6-phosphogluconate to 14CO2 (ref. 6). Thyroid slices were incubated and homogenized as above and the homogenate incubated for i h at 37 ° to allow for the destructi~m of the endogenous TPN. Homogenate representing approx. 2o mg of thyroid tissue was then incubated at 37 ° for 45 min in 1. 4 ml of buffer containing 25o/~moles of glycylglycine (pH 7.5), 15/~moles magnesium chloride and o.oi/zC (13ooo counts/rain) of [i-~4C]-6-phosphogluconate (specific activity 27.8 mC/mmole) and varying amounts of ATP and DPN. Incubations were terminated by the addition of o.2 nil of 3 M sulfuric acid to the incubation medium and o.5 ml of hyamine to the center well. x4CO2 was obtained and counted as previously described '~. Subcellular localization of the TSH-mediated increase in TPN was studied as follows. Approx. 2 g of thyroid slices were incubated in IO ml of Krebs-Ringer bicarbonate buffer for 45 rain in the presence and absence of TSH (o. 5 units/ml). The slices were then homogenized in IO ml of o.25 M sucrose containing o.o5 M nicotinamide and I ml of the homogenate was removed and extracted for TPN determination. The remaining homogenate was layered on io nfl of o.34 M sucrose-o.o5 M nicotinamide and centrifuged io min at 2ooo rev./min. The nuclear precipitate was resuspended in 5 ml of o.25 M sucrose-o.o5 M nicotinamide and recentrifuged for IO min. The nuclei were resuspended in 2 ml of water and I ml was extracted for TPN determination. The supernatant from the nuclei was centrifuged at ~2ooo :~ g for 2o rain Biochim. Biophys. Acla, Izr (I966) 241-249
TSH
EFFECTS
ON THYROID
DPN
243
KINASE
in a Servall centrifuge. The mitochondria were resuspended in 4 ml of 0.25 M sucrose0.05 M nicotinamide and recentrifuged. The mitochondrial button was suspended in I.I ml of water and i ml was extracted for TPN determination. The supernatant was centrifuged in a Spinco Model L ultracentrifuge at IOOOOO × g for I h. The microsomal pellet was resuspended in 1.2 ml of water and i ml of this and the remaining supernatant were extracted for TPN measurement. The subcellular localization of DPN kinase in thyroid was studied using a similar fractionation procedure. Aliquots of the various subcellular fractions were added to 1.2 ml of buffer containing 500/,moles of Tris (pH 7,5), 3 °/~moles of nicotinamide and I/,mole magnesium chloride. Appropriate tubes also contained o.16/~mole of ATP and 0.38 /,mole of DPN. After an incubation for 2 h at 37 ° without shaking, I ml was removed and extracted for TPN determination. TPN was measured as previously described s. Incorporation of E8-14Cladenine into nucleic acids was measured as follows. Thyroid slices were incubated at 37 ° with 1.25/zC (1250000 counts/min) ~8-1*C~adenine (specific activity 15 mC/Inmole) in 2 ml of Krebs-Ringer bicarbonate buffer without glucose for 45 rain. Appropriate flasks contained 0.05 mg of actinomycin D. At the end of the incubation, incorporation of [8-14C~adenine into nucleic acids was determined by the procedure of WOOL7. Control slices incubated for only 30 sec were included in the experiment. The isolated nucleic acids were dissolved in I ml of hyamine and counted in a liquid scintillation spectrometer using toluene containing 0.4% PPO and o.oi % POPOP as the phosphor. TSH, I unit per mg and 4 units per mg were gifts from Drs. ROBERT BATES and PETER C O N D L I F F E , National Institutes of Health and The Endocrine Study Section, National Institutes of Health, respectively. Comparable results were obtained using both of these preparations. Actinomycin D was kindly supplied by Dr. VINCENT ALBO, Children's Hospital, University of Pittsburgh. Vasopressin (I ml containing I0 pressor units) was obtained from Sandoz Pharmaceutical Corporation. EI-14C~Glucose and EB-14C~adenine were purchased from Nuclear-Chicago. [I-14C~-6Phosphogluconate was prepared as previously described 6. ATP and DPN were purchased from Sigma Chemical Company. DPN was purified by column chromatography by the procedure of DALZIELs. TABLE
I
SUBCELLULAR LOCALIZATION OF T S H
INDUCED INCREASE I ~ T P N
2 g o f t h y r o i d slices w e r e i n c u b a t e d i n i o m l o f K r e b s - R i n g e r bicarbonate buffer with and without T S H (o. 5 u n i t s / m l ) f o r 45 m i n . T h e slices w e r e t h e n h o m o g e n i z e d a n d f r a c t i o n a t e d a s d e s c r i b e d u n d e r EXPERIMENTAL PROCEDURE. T h e T P N c o n c e n t r a t i o n of e a c h f r a c t i o n w a s d e t e r m i n e d in triplicate.
Cell fraction
Whole homogenate Nuclei Mitochondria Microsomes Supernatant
T P N (re#moles/g) Control
TSH
8. 5 i .9 o. 8 o. I 7.o
23.6 2.4 o. 7 o. I 21.3
Biochim. Biophys. Acta, I Z i (1966) 2 4 1 - 2 4 9
244
j.B. FIELD et al.
RESULTS
The data in Table I indicate that almost all of the TSH stimulated TPN was in the supernatant portion of the cell. The TPN in the nuclear fraction was also slightly increased by TSH. Although an effect of TSH in a cell-free system could not be demonstrated, the data tabulated in Table II demonstrate that homogenates obtained from slices incubated with TSH had higher concentrations of TPN when incuTABLE II E F F E C T OF P R I O R I N C U B A T I O N OF T H Y R O I D ACTIVITY ASSAYED BY TPN FORMATION
SLICES
WITH
AND
WITHOUT
T S H ON D P N
KINASE
Aporox. 9o0 mg of thyroid slices were incubated with and w i t h o u t T S H (o.5 units). At the termination of 45 min incubation the slices were homogenized and T P N determined in an aliquot. Aliquots of the h o m o g e n a t e were incubated for 90 min in 2. 3 iiit of buffer containing 5o # m o l e s Tris (pH 7-5), 5 °/~inoles nicotinamide and 5 l*moles MgCI.~. o.16/~mole ATP and o. 15/tiiiole D P N were added to a p p r o p r i a t e tubes. T P N was m e a s u r e d in triplicate.
T P N (rnl, moles/g ) A t end of slices incubation
A t end of hornogenate incubation . . . . . . . . . -ATP --DPN +ATP+DPN
9.6 19.8
3.2 3.I
5.6 7.4
2.9 17. 4
o.9 i. 7
2.5 0.~
13.5 33-5
3.7 6.8
3.6 8.9
Expt. I Control TSH Expt. 2 Control TSH Expt. 3 Control TSH
T A B L E II1 EFFECT
OF
PRIOR
ACTIVITY ASSAYED
INCUBATION
OF
BY OXIDATION
THYROID OF
SLICES
V~*ITFI A N D
WITHOUT
~I-14C~-6-PHOSPHOGLUCONATE
TO
T S H ON D P N
KINASE
14CO 2
4oo mg of t h y r o i d slices were incubated for i h with and w i t h o u t TSH (o. 5 units/ml). A ~o% h o m o g e n a t e in w a t e r was p r e p a r e d and incubated at 37 ° for i h. H o m o g e n a t e equivalent to 2o mg of t h y r o i d was t h e n i n c u b a t e d in 1. 4 ml containing 25 ° #moles glycylglycine (pH 7.5), 15/tmoles MgCle, o.oi ~C [I-a4C]-6-phosphogluconate (13ooo counts/rain) and the a p p r o p r i a t e a m o u n t s of D P N and ATP. This i n c u b a t i o n was 45 rain. The results are the m e a n s ± S.E. of the m e a n of triplicate d e t e r m i n a t i o n s and expressed as c o u n t s / m i n per 2o m g of t h y r o i d h o m o genate.
I~tcubation conditions
14C02 counts/rnin Control
DPN,
--
0.02/~mole 0.08 l, mole o.38/*mole o.38 #mole
AT P DPN, DPN, DPN, DPN,
0. 4 / , n I o l e ATP 0. 4/*mole ATP --ATP o.I p m o l e ATP
265 I385 3561 486i 5913
Biochirn. tliophys. Acta, I21 (:966) 24I 249
) ~ ~ ~ ~
TSH 28 89 256 2o 4 207
314 ~ 20 I764 ~ 34 5267 ~ 209 6757 ~ 218 7 4 0 6 : 2 525
T S H EFFECTS ON THYROID DPN KII~ASE
245
bated with ATP and DPN than did homogenates prepared from control slices. At the termination of the slice incubation, T P N levels were higher in the presence of TSH but during subsequent homogenate incubation in the absence of ATP and DPN there was a marked reduction in T P N content so that they were usually comparable to those in the homogenate obtained from the control slices. However, in the presence of ATP and DPN, the T P N levels were almost always higher and this increase was greater in homogenates made from TSH incubated slices. New synthesis of T P N was not documented since the T P N levels in homogenates incubated with ATP and DPN were lower than the corresponding levels in slices just prior to homogenization. In an attempt to detect T P N as it was being formed, experiments were performed measuring T P N indirectly as a function of 14C02 production from ~I-14C1-6phosphogluconate in the presence and absence of ATP and DPN (Table III). Prior to incubation with and without ATP and DPN the homogenates were kept at 37 ° for i h which was sufficient to reduce the T P N values to very low levels as reflected by low 14CO2 counts. When varying amounts of DPN and ATP were added, more ~i-14C]-6-phosphogluconate was oxidized by the homogenate prepared from TSH incubated slices. Although this represents an indirect method of measuring T P N and thus DPN kinase activity, previous studies have shown that glucose oxidation in thyroid homogenate was dependent upon T P N concentrations °. The data in Table IV T A B L E IV
*
RELATIONSHIP BETWEEN AMOUNT OF THYROID HOMOGENATE AND D P N KINASE ACTIVITYASSAYED BY OXIDATION OF [I-14C~-6-PHOSPHOGLUCONATE
3oo mg of t h y r o i d slices were incubated for I h with and w i t h o u t T S H (0. 5 units/ml). A io % h o m o g e n a t e in w a t e r was p r e p a r e d and incubated at 37 ° for i h. Aliquots of the h o m o g e n a t e were t h e n incubated for 45 min in 1. 4 ml containing 250 /*moles glycylglycine (pH 7.5), 15/~moles MgC12, o.oi ~uC EI-14C]-6-phosphogluconate (13000 counts/min) and 0.38 #mole D P N in the a p p r o p r i a t e tubes. The results are the m e a n s ± S.E. of the m e a n of triplicate determinations and expressed as c o u n t s / m i n per a m o u n t of t h y r o i d incubated.
Homogenate equivalent to amount of thyroid
14C0~ counts/rain
Control --ATP, - - D P N
7.5mg 15 mg 30 mg
86 ~ 13 143 ~ 27 2Ol ~ 26
TSH +ATP, +DPN 386 ± 14 654 ~ lO9 136o i 88
--ATP, - - D P N 86 ± 23 91 i 44 145 ± 5
+ATP, +DPN 395 • IO 858 ± 39 184o ± 2Ol
demonstrate that oxidation of ~i-14C2-6-phosphogluconate was proportional to the amount of thyroid homogenate. Addition of partially purified 6-phosphogluconic dehydrogenase (6-phospho-D-gluconate:TPN + oxidoreductase, EC 1.1.1.44 ) to the homogenate did not increase 14CO~ indicating that this enzyme was not limiting the reaction (unpublished observations). Subcellular fractionation of thyroid homogenate indicated that the highest concentration of DPN kinase was in the nuclear fraction (Table V). The data in Table VI indicate that actinomycin D did not modify either basal glucose oxidation or the T S H stimulation even though E8-14C~adenine incorporation Biochim. Biophys. Acta, 121 (1966) 241-249
246
j.B. FIELD et al.
TABLE V SUBCELLULAR LOCALIZATION OF THYROID DPN
K I N A S E A S S A Y E D BY T P N
FORMATION
2 g of t h y r o i d were h o m o g e n i z e d a n d f r a c t i o n a t e d as d e s c r i b e d u n d e r EXPERIMENTAL PROCEDURE. A l i q u o t s of t h e wh ole h o m o g e n a t e a n d s u b c e l l u l a r f r a c t i o n s were i n c u b a t e d in 1.2 nil c o n t a i n i n g 5 ° / * m o l e s Tris (p H 7.5), 3 ° / * m o l e s n i c o t i n a m i d e a n d I t t mol e MgCl 2. o.16 l~naole A T P a nd 0 . 3 8 / * m o l e D P N were a d d e d to t h e a p p r o p r i a t e tubes , T P N w a s d e t e r m i n e d in t r i p l i c a t e a f t e r a 2-tl i n c u b a t i o n . The r e s u l t s are e x p r e s s e d as T P N f o r m a t i o n p e r g of o r i g i n a l t h y r o i d tissue.
Cell fraction
T P N (ml, moles/g ) -ATP,
Whole homogenate Nuclei Mitochondria Microsomes Supernatant
DPN
o.23 o.o6 o.o 7 o.o 3 o.o8
+ATP, +DPN o.8o o.28 o. 12 o.o8 o. I I
TABLE VI E F F E C T OF A C T I N O M Y C I N D ON T S H S T I M U L A T I O N O F G L U C O S E O X I D A T I O N A N D E S - 1 4 C ] A D E N I N E I N C O R P O R A T I O N I N T O I~N~A I N T H Y R O I D S L I C E S
Glucose c o n c e n t r a t i o n was 0. 7 m g / m l a n d 250000 c o u n t s / m i n [I-laCjglueose in e a c h fl a s k w h e n 14CO~ p r o d u c t i o n w a s m e a s u r e d . I n c u b a t i o n s were for 45 rain. N o glucose w a s a d d e d to t h e flasks w h e n [8-14C]adenine (1.25 tzC a n d 1 2 5 0 0 0 0 c o u n t s / r a i n ) i n c o r p o r a t i o n i n t o n u c l e i c a c i ds w a s s t u d i e d . Slices i n c u b a t e d for 3 ° sec were also i n c l u d e d in t h i s p a r t of t h e e x p e r i m e n t t o c o r r e c t for r a d i o a c t i v i t y w h i c h n l a y h a v e b e e n c o p r e c i p i t a t e d w i t h t h e n u c l e i c acids. The r e s u l t s are m e a n s ± S.E. of t h e m e a n of t r i p l i c a t e d e t e r m i n a t i o n s a n d e x p r e s s e d as c o u n t s / r a i n pe r g t h y r o i d p e r 45-rain i n c u b a t i o n .
Substance
Amount per flask
1~C02 (counts/rnin per g)
RNA (counts/rain per g)
Control Actinomycin D TSH Actinomycin, TSH
0.05 m g 0. 5 u n i t s 0.05 rag, 0. 5 u n i t s
19 22 51 47
5894 ~: 1876 578 7{: 29
233 633 567 7° 0
± ~ ± ±
lO41 144o 1613 3234
TABLE VII I~FFI~CT i1¢ vitro OF T S H SALIVARY GLAND SLICES
ON [ I - 1 4 C ] G L U C O S E O X I D A T I O N B Y D O G T H Y R O I D , G A S T R I C M U C O S A , A N D
Glucose c o n c e n t r a t i o n w a s I m g / m l a n d 0.25/~C (25oooo c o u n t s / r a i n ) [I-14Qglucose w a s p r e s e n t in each flask. The r e s u l t s aTe t h e m e a n s .+_ S.E. of t h e m e a n of t r i p l i c a t e d e t e r m i n a t i o n s a n d exp r e s s e d as c o u n t s / m i n p e r g t i s s u e p e r 45-rain i n c u b a t i o n .
Tissue
TSH (0.5 units)
14C0 2 (counts/min per g)
Thyroid Thyroid Gastric mucosa Gastric mucosa Submaxillary gland Submaxillary gland
+ + -+
io 36 7 5 18 17
500 15o 967 233 ooo 933
Biochim. Biophys. Acta, I 2 i (1966) 241 249
± -k ~ •
1349 899 2556 633
TSH
EFFECTS ON THYROID D P N
KINASE
247
into nucleic acids was reduced b y 9 ° %. Although gastric mucosa 1° and submandibular gland 11 share with thyroid the ability to concentrate iodide, glucose oxidation in the former two was not stimulated by TSH. Evidence for a direct stimulatory effect of vasopressin on thyroid gland function has been presentedl*, 13. However, the data in Table V I I I indicate that vasopressin did not reproduce the effects of T S H on glucose oxidation. In fact vasopressin, at a dosage which had no demonstrable effect on basal glucose oxidation, partially inhibited the TSH stimulation. TABLE VIII EFFECT
i~ vitro
OF VASOPRESSIN ON THYROID GLUCOSE OXIDATION AND
TSH
RESPONSIVENESS
Glucose c o n c e n t r a t i o n was o.7 m g / m l a n d 0.25 pC (25oooo counts/rain) [I-14C]glucose was p r e s e n t in each flask. T h e results are m e a n s ~ S.E. of t h e m e a n of triplicate d e t e r m i n a t i o n s a n d e x p r e s s e d as c o u n t s / m i n p e r g t h y r o i d t i s s u e p e r 45-rain i n c u b a t i o n .
Substance
Amount per flask
14C02 (counts/min per g)
Control
--
13 442 ± 287
Vasopressin
2
TSH
o.25 u n i t s
Vasopressin, T S H
2
units
12 781 :~ 892 31 704 ~ 1812
units, 0.25 u n i t s 21 791 :~ 469
DISCUSSION
The observation that T S H increased T P N levels in thyroid slices and caused an equivalent decrease in DPN levels suggested an effect of the hormone on the enzyme DPN kinase which converts ATP and DPN to T P N (refs. I, 2). Since the addition of TSH to thyroid homogenates had no effect on DPN kinase activity, this enzyme was measured in homogenates prepared from slices incubated with and without TSH. Although the data in Table I I indicate that homogenates obtained from TSH-treated slices and incubated with ATP and DPN contained more T P N than did homogenates prepared from control slices, it does not necessarily signify augmented D P N kinase activity since net T P N synthesis was not obtained. The addition of A T P and D P N was associated with higher levels of T P N in the homogenate prepared from the TSH exposed slices, but they were still consistently less than the amount present at the end of the slice incubation. The results obtained could be equally well explained by an inhibition of destruction of T P N in the presence of ATP and D P N by the homogenate prepared from slices incubated with TSH. Since these experiments did not discriminate between the possibility of increased T P N synthesis and inhibition of T P N degradation, studies were done in which "~PN formation was measured in terms of [I-14CJ-6-phosphogluconate oxidation to 14CO~. In such a system T P N might be utilized for oxidation of [I-14C]-6-phosphogluconate before being destroyed. In this system the evolution of 14CO2 was not limited by 6-phosphogluconic dehydrogenase and was related to the amount of T P N present (unpublished observations). To correct for the initially higher T P N in the homogenate obtained from T S H incubated slices homogenates were kept at 37 ° for Biochim. Biophys. Acta, 121 (1966) 241-249
24S
j.B. WELD et al.
I h in the absence of nicotinamide. At the end of this time endogenous TPN was very low in both homogenates (Table III). Addition of various amounts of ATP and DPN increased the oxidation of [I-~4C~-6-phosphogluconate and at any level of ATP and DPN the increment was greater using homogenate obtained from the TSH incubated slices. These results are consistent with net TPN synthesis and increased DPN kinase activity in homogenate prepared from slices incubated with TSH. The data indicate that the concentration of DPN influenced 6-phosphogluconate oxidation more than that of ATP. This observation is in contrast to our previous finding that moderate reduction of DPN in thyroid slices by incubation with Carzinophilin did not inhibit the T S H effect on either glucose oxidation or increased TPN synthesis 4. t;urthermore the importance of ATP in the T S H effect on thyroid slices is suggested by the inhibition of TSH stimulation by metabolic inhibitors which presumably act by impairing ATP generation 14. One possible explanation for this apparent discrepancy between slice and homogenate results is that endogenous ATP in homogenates is present in excess and the rate of destruction of DPN is accelerated to the point where it becomes rate limiting. Although these experiments demonstrate an effect of TSH on DPN kinase activity, they do not elucidate the importance of intracellular concentrations of ATP and DPN in respect to TPN synthesis in intact thyroid cells. Most of the DPN kinase activity was localized in the nuclear fraction contrary to the results of CLAI~Kel al. '~ who reported the activity in the supernatant of liver cells. The TSH-mediated increase in TPN in thyroid slices was found in the supernatant (Table I). Such localization could represent leakage of TPN into the supernatant from another subcellular component during fractionation of the cells. The nuclear localization of DPN kinase would support this possibility. Since previous results had demonstrated that the TSH stimulation was independent of new protein synthesis°- it was not suprising that actinomycin D which inhibits RNA synthesis t6 did not modify the augmented glucose oxidation of thyroid slices exposed to T S H (Table VI). The concentration used was sufficient to inhibit ~8J~C]adenine incorporation into nucleic acids by 9 ° °o yet it did not influence basal or hormonal stimulation of glucose oxidation. Although it is currently fashionable to implicate RNA synthesis in hormone action '7 it is apparent that effects of TSH on glucose oxidation and TPN synthesis can not be explained by such a mechanism. The thyroid gland shares with gastric mucosa and salivary gland the ability to concentrate iodide. However, the failure of TSH to influence glucose oxidation in gastric mucosa and submandibular gland is consistent with its inability to stimulate iodide concentration in these tissues. Indirect evidence has been presented suggesting a direct stimulatorv effect of vasopressin on thyroid gland function12,13. If vasopressin does mimic TSH in stimulating thyroxine release it does not do so as a result of increasing glucose oxidation (Table VIII). An amount of vasopressin which by itself had no effect on basal glucose oxidation actually partially inhibited the action of TSH. The mechanism of such inhibition is not evident at present. ACKNOWLEDGEMENT
This investigation was supported by U.S. Public Health Service Grant AMo6865 from the National Institutes of Health. Biochim. Biophys. Acta, 121 (I966) 241- 249
TSH
EFFECTS ON THYROID D P N
KINASE
249
REFERENCES i 2 3 4 5 6 7 8 9 IO 11 12 13 14 15 16 17
I. PASTAN, B. HERRING AND J. B. FIELD, J. Biol. Chem., 236 (1961) PC 25. J. B. FIELD, P. JOHNSON, E. KENDIG AND I. PASTAN, J. Biol. Chem., 238 (1963) 1189. 1. PASTAN, P. JOHNSON, E. KENDIG AND J. B. FIELD, J. Biol. Chem., 238 (1963) 3366. J. B. FIELD, A. K. REMER AND S. M. EPSTEIN, J. Biol. Chem., 240 (1965) 883. J. B. FIELD, I. PASTAN, P. JOHNSON AND B. HERRING, J. Biol. Chem., z35 (196o) 1863. I. PASTAN, V. WILLS, B. HERRING AND J. B. FIELD, J. Biol. Chem., 238 (1963) 3362. I. G. WooL, Biochim. Biophys. Acta, 68 (I963) 28. K. DALZlEL, jr. Biol. Chem., 238 (1963) 1538. J. B. FIELD, I. PASTAN, B. HERRING AND P. JOHNSON, Biochim. Biophys. Acta, 5o (1961) 513 . ~T. S. HALMI, Ciba Foundation Colloquia on Endocrinology, io (1956) 79A. TAUROG, G. D, POTTER AND I. L. CHAIKOFF, Endocrinology, 64 (1959) lO38. G. FEUER, Nature, 197 (1963) 1176. C. Y. SOWERS, T. W. REDDING AND A. V. SCHALLY, Endocrinology, 74 (1964) 559. B. W. O'MALLE¥ AND J. B. FIELD, Biochim. Biophys. Acta, 9o (1964) 349. J. B. CLARK, A. L. GREENBAUM, P. McLEAN AND E. REID, Nature, 2Ol (1964) I I 3 I . E. REICH, R. M. FRANKLIN, A. J. SHATKIN AND E. L. TATUM, Science, 134 (1961 ~ 556. L. D. SAMUELS, New EnglandJ. Med., 271 (1964) 13Ol.
Biochim. Biophys. Acta, 121 (1966) 241-249
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
25564
P Y R I D I N E NUCLEOTIDES IN T H E THYROID VI. DPN KINASE ACTIVITY OF THYROID HOMOGENATE OBTAINED FROM SLICES INCUBATED W I T H AND WITHOUT THYROID STIMULATING HORMONE J A M E S B. F I E L D , S H E L D O N M. E P S T E I N * , A D R I E N N E BOYLE
K. R E M E R
AND C O N S T A N C E
The Clinical Research Unit and the Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. (U.S.A.) (Received O c t o b e r 29th, 1965)
SUMMARY
Thyroid slices incubated with and without TSH have been homogenized and assayed for DPN kinase (ATP:DPN 2'-phosphotransferase, EC 2.7.1.23) activity. Such activity was increased when homogenates from TSH incubated slices were incubated with ATP and DPN and [i-14C]-6-phosphogluconate oxidation measured as an indication of TPN formation. In similar experiments when TPN was measured directly, homogenates obtained from TSH incubated slices also had greater TPN levels than homogenates prepared from control slices. However, in these experiments since net synthesis of TPN was not observed, the results could also be explained by decreased rate of destruction as well as by increased synthesis of TPN. Subcellular fractionation of thyroid slices localized most of the DPN kinase to the nuclear fraction while the increased TPN of TSH incubated slices was in the supernatant. The TSH stimulation of glucose oxidation was not inhibited b y a concentration of actinomycin D which caused a 9 ° % reduction in [SA*C]adenine incorporation into nucleic acids. Vasopressin, which has been reported to directly stimulate thyroxine release from the thyroid, did not stimulate glucose oxidation in thyroid slices. TSH did not influence glucose oxidation of gastric mucosa and submandibular gland, two tissues which also share with thyroid the ability to concentration iodide but are not TSH responsive in terms of this parameter.
INTRODUCTION
The stimulation of glucose oxidation in thyroid slices induced by TSH has been attributed to the increased TPN observed in such slices1, 2. The increased TPN was associated with an equivalent decrease in DPN (ref. I) and could not be accounted for by changes in T P N H (ref. 3). These results suggested an effect of TSH on the enzyme DPN kinase (ATP:DPN 2'-phosphotransferase, EC 2.7.1.23) which converts * P o s t d o c t o r a l Fellow, U.S. P u b l i c H e a l t h Service.
Biochim. Biophys. Acta, 121 (1966) 241-249
242
J.B. FIELD et al.
DPN and ATP to TPN. New protein synthesis apparently was not involved since puromycin did not inhibit responsiveness to TSH although incorporation of [i-t4C!leucine into protein was reduced 98 % (ref. 2). Moderate reductions of thyroidal DPN induced by Carzinophilin did not modify TSH stimulation of glucose oxidation or TPN synthesis 4 indicating that DPN was not limiting in TPN synthesis and that TSH did not act by influencing DPN levels. The present studies were undertaken to assess the effects of TSH on the thyroid gland DPN kinase reaction in hopes of further elucidating the mechanism of action of TSH. EXPERIMENTAL PROCEDURE
The method of preparation and incubation of thyroid slices has been described 5. Thegastric mucosae were separated from the rest of the stomach by blunt dissection and sliced with a Stadie-Riggs microtome. The effect of prior incubation of thyroid slices with TSH on the subsequent activity of DPN kinase in homogenates was studied as follows. Approx. 9oo mg of thyroid slices were incubated in IO ml of Krebs-Ringer bicarbonate buffer with and without TSH (o.5 units/ml) for 45 min. Following incubation a Io % homogenate in glass-distilled water was prepared, i ml of each homogenate was extracted immediately in 3 ml of o.i M HC1 to determine the TPN concentration at the end of the slice incubation. I ml of the homogenate obtained from the control or TSH incubated slices was incubated for 9 ° min at 37 in 2.3 ml of buffer containing 50/~moles of Tris (pH 7.5), 5o/,moles nicotinamide, 5/~moles of magnesium chloride, and in appropriate tubes, o.I 5/,mole of DPN and o.16/~mole ATP. Controls without homogenate but with DPN and ATP were included in each experiment. At the end of the incubation, the reaction mixture was extracted with 2 ml of o.I M HC1 at IOO° for 3o see for TPN measurement. The effects of incubation of thyroid slices with TSH on DPN kinase activity of homogenates was also assessed by an indirect measurement of TPN based on the oxidation of [I-i4(; 6-phosphogluconate to 14CO2 (ref. 6). Thyroid slices were incubated and homogenized as above and the homogenate incubated for i h at 37 ° to allow for the destructi~m of the endogenous TPN. Homogenate representing approx. 2o mg of thyroid tissue was then incubated at 37 ° for 45 min in 1. 4 ml of buffer containing 25o/~moles of glycylglycine (pH 7.5), 15/~moles magnesium chloride and o.oi/zC (13ooo counts/rain) of [i-~4C]-6-phosphogluconate (specific activity 27.8 mC/mmole) and varying amounts of ATP and DPN. Incubations were terminated by the addition of o.2 nil of 3 M sulfuric acid to the incubation medium and o.5 ml of hyamine to the center well. x4CO2 was obtained and counted as previously described '~. Subcellular localization of the TSH-mediated increase in TPN was studied as follows. Approx. 2 g of thyroid slices were incubated in IO ml of Krebs-Ringer bicarbonate buffer for 45 rain in the presence and absence of TSH (o. 5 units/ml). The slices were then homogenized in IO ml of o.25 M sucrose containing o.o5 M nicotinamide and I ml of the homogenate was removed and extracted for TPN determination. The remaining homogenate was layered on io nfl of o.34 M sucrose-o.o5 M nicotinamide and centrifuged io min at 2ooo rev./min. The nuclear precipitate was resuspended in 5 ml of o.25 M sucrose-o.o5 M nicotinamide and recentrifuged for IO min. The nuclei were resuspended in 2 ml of water and I ml was extracted for TPN determination. The supernatant from the nuclei was centrifuged at ~2ooo :~ g for 2o rain Biochim. Biophys. Acla, Izr (I966) 241-249
TSH
EFFECTS
ON THYROID
DPN
243
KINASE
in a Servall centrifuge. The mitochondria were resuspended in 4 ml of 0.25 M sucrose0.05 M nicotinamide and recentrifuged. The mitochondrial button was suspended in I.I ml of water and i ml was extracted for TPN determination. The supernatant was centrifuged in a Spinco Model L ultracentrifuge at IOOOOO × g for I h. The microsomal pellet was resuspended in 1.2 ml of water and i ml of this and the remaining supernatant were extracted for TPN measurement. The subcellular localization of DPN kinase in thyroid was studied using a similar fractionation procedure. Aliquots of the various subcellular fractions were added to 1.2 ml of buffer containing 500/,moles of Tris (pH 7,5), 3 °/~moles of nicotinamide and I/,mole magnesium chloride. Appropriate tubes also contained o.16/~mole of ATP and 0.38 /,mole of DPN. After an incubation for 2 h at 37 ° without shaking, I ml was removed and extracted for TPN determination. TPN was measured as previously described s. Incorporation of E8-14Cladenine into nucleic acids was measured as follows. Thyroid slices were incubated at 37 ° with 1.25/zC (1250000 counts/min) ~8-1*C~adenine (specific activity 15 mC/Inmole) in 2 ml of Krebs-Ringer bicarbonate buffer without glucose for 45 rain. Appropriate flasks contained 0.05 mg of actinomycin D. At the end of the incubation, incorporation of [8-14C~adenine into nucleic acids was determined by the procedure of WOOL7. Control slices incubated for only 30 sec were included in the experiment. The isolated nucleic acids were dissolved in I ml of hyamine and counted in a liquid scintillation spectrometer using toluene containing 0.4% PPO and o.oi % POPOP as the phosphor. TSH, I unit per mg and 4 units per mg were gifts from Drs. ROBERT BATES and PETER C O N D L I F F E , National Institutes of Health and The Endocrine Study Section, National Institutes of Health, respectively. Comparable results were obtained using both of these preparations. Actinomycin D was kindly supplied by Dr. VINCENT ALBO, Children's Hospital, University of Pittsburgh. Vasopressin (I ml containing I0 pressor units) was obtained from Sandoz Pharmaceutical Corporation. EI-14C~Glucose and EB-14C~adenine were purchased from Nuclear-Chicago. [I-14C~-6Phosphogluconate was prepared as previously described 6. ATP and DPN were purchased from Sigma Chemical Company. DPN was purified by column chromatography by the procedure of DALZIELs. TABLE
I
SUBCELLULAR LOCALIZATION OF T S H
INDUCED INCREASE I ~ T P N
2 g o f t h y r o i d slices w e r e i n c u b a t e d i n i o m l o f K r e b s - R i n g e r bicarbonate buffer with and without T S H (o. 5 u n i t s / m l ) f o r 45 m i n . T h e slices w e r e t h e n h o m o g e n i z e d a n d f r a c t i o n a t e d a s d e s c r i b e d u n d e r EXPERIMENTAL PROCEDURE. T h e T P N c o n c e n t r a t i o n of e a c h f r a c t i o n w a s d e t e r m i n e d in triplicate.
Cell fraction
Whole homogenate Nuclei Mitochondria Microsomes Supernatant
T P N (re#moles/g) Control
TSH
8. 5 i .9 o. 8 o. I 7.o
23.6 2.4 o. 7 o. I 21.3
Biochim. Biophys. Acta, I Z i (1966) 2 4 1 - 2 4 9
244
j.B. FIELD et al.
RESULTS
The data in Table I indicate that almost all of the TSH stimulated TPN was in the supernatant portion of the cell. The TPN in the nuclear fraction was also slightly increased by TSH. Although an effect of TSH in a cell-free system could not be demonstrated, the data tabulated in Table II demonstrate that homogenates obtained from slices incubated with TSH had higher concentrations of TPN when incuTABLE II E F F E C T OF P R I O R I N C U B A T I O N OF T H Y R O I D ACTIVITY ASSAYED BY TPN FORMATION
SLICES
WITH
AND
WITHOUT
T S H ON D P N
KINASE
Aporox. 9o0 mg of thyroid slices were incubated with and w i t h o u t T S H (o.5 units). At the termination of 45 min incubation the slices were homogenized and T P N determined in an aliquot. Aliquots of the h o m o g e n a t e were incubated for 90 min in 2. 3 iiit of buffer containing 5o # m o l e s Tris (pH 7-5), 5 °/~inoles nicotinamide and 5 l*moles MgCI.~. o.16/~mole ATP and o. 15/tiiiole D P N were added to a p p r o p r i a t e tubes. T P N was m e a s u r e d in triplicate.
T P N (rnl, moles/g ) A t end of slices incubation
A t end of hornogenate incubation . . . . . . . . . -ATP --DPN +ATP+DPN
9.6 19.8
3.2 3.I
5.6 7.4
2.9 17. 4
o.9 i. 7
2.5 0.~
13.5 33-5
3.7 6.8
3.6 8.9
Expt. I Control TSH Expt. 2 Control TSH Expt. 3 Control TSH
T A B L E II1 EFFECT
OF
PRIOR
ACTIVITY ASSAYED
INCUBATION
OF
BY OXIDATION
THYROID OF
SLICES
V~*ITFI A N D
WITHOUT
~I-14C~-6-PHOSPHOGLUCONATE
TO
T S H ON D P N
KINASE
14CO 2
4oo mg of t h y r o i d slices were incubated for i h with and w i t h o u t TSH (o. 5 units/ml). A ~o% h o m o g e n a t e in w a t e r was p r e p a r e d and incubated at 37 ° for i h. H o m o g e n a t e equivalent to 2o mg of t h y r o i d was t h e n i n c u b a t e d in 1. 4 ml containing 25 ° #moles glycylglycine (pH 7.5), 15/tmoles MgCle, o.oi ~C [I-a4C]-6-phosphogluconate (13ooo counts/rain) and the a p p r o p r i a t e a m o u n t s of D P N and ATP. This i n c u b a t i o n was 45 rain. The results are the m e a n s ± S.E. of the m e a n of triplicate d e t e r m i n a t i o n s and expressed as c o u n t s / m i n per 2o m g of t h y r o i d h o m o genate.
I~tcubation conditions
14C02 counts/rnin Control
DPN,
--
0.02/~mole 0.08 l, mole o.38/*mole o.38 #mole
AT P DPN, DPN, DPN, DPN,
0. 4 / , n I o l e ATP 0. 4/*mole ATP --ATP o.I p m o l e ATP
265 I385 3561 486i 5913
Biochirn. tliophys. Acta, I21 (:966) 24I 249
) ~ ~ ~ ~
TSH 28 89 256 2o 4 207
314 ~ 20 I764 ~ 34 5267 ~ 209 6757 ~ 218 7 4 0 6 : 2 525
T S H EFFECTS ON THYROID DPN KII~ASE
245
bated with ATP and DPN than did homogenates prepared from control slices. At the termination of the slice incubation, T P N levels were higher in the presence of TSH but during subsequent homogenate incubation in the absence of ATP and DPN there was a marked reduction in T P N content so that they were usually comparable to those in the homogenate obtained from the control slices. However, in the presence of ATP and DPN, the T P N levels were almost always higher and this increase was greater in homogenates made from TSH incubated slices. New synthesis of T P N was not documented since the T P N levels in homogenates incubated with ATP and DPN were lower than the corresponding levels in slices just prior to homogenization. In an attempt to detect T P N as it was being formed, experiments were performed measuring T P N indirectly as a function of 14C02 production from ~I-14C1-6phosphogluconate in the presence and absence of ATP and DPN (Table III). Prior to incubation with and without ATP and DPN the homogenates were kept at 37 ° for i h which was sufficient to reduce the T P N values to very low levels as reflected by low 14CO2 counts. When varying amounts of DPN and ATP were added, more ~i-14C]-6-phosphogluconate was oxidized by the homogenate prepared from TSH incubated slices. Although this represents an indirect method of measuring T P N and thus DPN kinase activity, previous studies have shown that glucose oxidation in thyroid homogenate was dependent upon T P N concentrations °. The data in Table IV T A B L E IV
*
RELATIONSHIP BETWEEN AMOUNT OF THYROID HOMOGENATE AND D P N KINASE ACTIVITYASSAYED BY OXIDATION OF [I-14C~-6-PHOSPHOGLUCONATE
3oo mg of t h y r o i d slices were incubated for I h with and w i t h o u t T S H (0. 5 units/ml). A io % h o m o g e n a t e in w a t e r was p r e p a r e d and incubated at 37 ° for i h. Aliquots of the h o m o g e n a t e were t h e n incubated for 45 min in 1. 4 ml containing 250 /*moles glycylglycine (pH 7.5), 15/~moles MgC12, o.oi ~uC EI-14C]-6-phosphogluconate (13000 counts/min) and 0.38 #mole D P N in the a p p r o p r i a t e tubes. The results are the m e a n s ± S.E. of the m e a n of triplicate determinations and expressed as c o u n t s / m i n per a m o u n t of t h y r o i d incubated.
Homogenate equivalent to amount of thyroid
14C0~ counts/rain
Control --ATP, - - D P N
7.5mg 15 mg 30 mg
86 ~ 13 143 ~ 27 2Ol ~ 26
TSH +ATP, +DPN 386 ± 14 654 ~ lO9 136o i 88
--ATP, - - D P N 86 ± 23 91 i 44 145 ± 5
+ATP, +DPN 395 • IO 858 ± 39 184o ± 2Ol
demonstrate that oxidation of ~i-14C2-6-phosphogluconate was proportional to the amount of thyroid homogenate. Addition of partially purified 6-phosphogluconic dehydrogenase (6-phospho-D-gluconate:TPN + oxidoreductase, EC 1.1.1.44 ) to the homogenate did not increase 14CO~ indicating that this enzyme was not limiting the reaction (unpublished observations). Subcellular fractionation of thyroid homogenate indicated that the highest concentration of DPN kinase was in the nuclear fraction (Table V). The data in Table VI indicate that actinomycin D did not modify either basal glucose oxidation or the T S H stimulation even though E8-14C~adenine incorporation Biochim. Biophys. Acta, 121 (1966) 241-249
246
j.B. FIELD et al.
TABLE V SUBCELLULAR LOCALIZATION OF THYROID DPN
K I N A S E A S S A Y E D BY T P N
FORMATION
2 g of t h y r o i d were h o m o g e n i z e d a n d f r a c t i o n a t e d as d e s c r i b e d u n d e r EXPERIMENTAL PROCEDURE. A l i q u o t s of t h e wh ole h o m o g e n a t e a n d s u b c e l l u l a r f r a c t i o n s were i n c u b a t e d in 1.2 nil c o n t a i n i n g 5 ° / * m o l e s Tris (p H 7.5), 3 ° / * m o l e s n i c o t i n a m i d e a n d I t t mol e MgCl 2. o.16 l~naole A T P a nd 0 . 3 8 / * m o l e D P N were a d d e d to t h e a p p r o p r i a t e tubes , T P N w a s d e t e r m i n e d in t r i p l i c a t e a f t e r a 2-tl i n c u b a t i o n . The r e s u l t s are e x p r e s s e d as T P N f o r m a t i o n p e r g of o r i g i n a l t h y r o i d tissue.
Cell fraction
T P N (ml, moles/g ) -ATP,
Whole homogenate Nuclei Mitochondria Microsomes Supernatant
DPN
o.23 o.o6 o.o 7 o.o 3 o.o8
+ATP, +DPN o.8o o.28 o. 12 o.o8 o. I I
TABLE VI E F F E C T OF A C T I N O M Y C I N D ON T S H S T I M U L A T I O N O F G L U C O S E O X I D A T I O N A N D E S - 1 4 C ] A D E N I N E I N C O R P O R A T I O N I N T O I~N~A I N T H Y R O I D S L I C E S
Glucose c o n c e n t r a t i o n was 0. 7 m g / m l a n d 250000 c o u n t s / m i n [I-laCjglueose in e a c h fl a s k w h e n 14CO~ p r o d u c t i o n w a s m e a s u r e d . I n c u b a t i o n s were for 45 rain. N o glucose w a s a d d e d to t h e flasks w h e n [8-14C]adenine (1.25 tzC a n d 1 2 5 0 0 0 0 c o u n t s / r a i n ) i n c o r p o r a t i o n i n t o n u c l e i c a c i ds w a s s t u d i e d . Slices i n c u b a t e d for 3 ° sec were also i n c l u d e d in t h i s p a r t of t h e e x p e r i m e n t t o c o r r e c t for r a d i o a c t i v i t y w h i c h n l a y h a v e b e e n c o p r e c i p i t a t e d w i t h t h e n u c l e i c acids. The r e s u l t s are m e a n s ± S.E. of t h e m e a n of t r i p l i c a t e d e t e r m i n a t i o n s a n d e x p r e s s e d as c o u n t s / r a i n pe r g t h y r o i d p e r 45-rain i n c u b a t i o n .
Substance
Amount per flask
1~C02 (counts/rnin per g)
RNA (counts/rain per g)
Control Actinomycin D TSH Actinomycin, TSH
0.05 m g 0. 5 u n i t s 0.05 rag, 0. 5 u n i t s
19 22 51 47
5894 ~: 1876 578 7{: 29
233 633 567 7° 0
± ~ ± ±
lO41 144o 1613 3234
TABLE VII I~FFI~CT i1¢ vitro OF T S H SALIVARY GLAND SLICES
ON [ I - 1 4 C ] G L U C O S E O X I D A T I O N B Y D O G T H Y R O I D , G A S T R I C M U C O S A , A N D
Glucose c o n c e n t r a t i o n w a s I m g / m l a n d 0.25/~C (25oooo c o u n t s / r a i n ) [I-14Qglucose w a s p r e s e n t in each flask. The r e s u l t s aTe t h e m e a n s .+_ S.E. of t h e m e a n of t r i p l i c a t e d e t e r m i n a t i o n s a n d exp r e s s e d as c o u n t s / m i n p e r g t i s s u e p e r 45-rain i n c u b a t i o n .
Tissue
TSH (0.5 units)
14C0 2 (counts/min per g)
Thyroid Thyroid Gastric mucosa Gastric mucosa Submaxillary gland Submaxillary gland
+ + -+
io 36 7 5 18 17
500 15o 967 233 ooo 933
Biochim. Biophys. Acta, I 2 i (1966) 241 249
± -k ~ •
1349 899 2556 633
TSH
EFFECTS ON THYROID D P N
KINASE
247
into nucleic acids was reduced b y 9 ° %. Although gastric mucosa 1° and submandibular gland 11 share with thyroid the ability to concentrate iodide, glucose oxidation in the former two was not stimulated by TSH. Evidence for a direct stimulatory effect of vasopressin on thyroid gland function has been presentedl*, 13. However, the data in Table V I I I indicate that vasopressin did not reproduce the effects of T S H on glucose oxidation. In fact vasopressin, at a dosage which had no demonstrable effect on basal glucose oxidation, partially inhibited the TSH stimulation. TABLE VIII EFFECT
i~ vitro
OF VASOPRESSIN ON THYROID GLUCOSE OXIDATION AND
TSH
RESPONSIVENESS
Glucose c o n c e n t r a t i o n was o.7 m g / m l a n d 0.25 pC (25oooo counts/rain) [I-14C]glucose was p r e s e n t in each flask. T h e results are m e a n s ~ S.E. of t h e m e a n of triplicate d e t e r m i n a t i o n s a n d e x p r e s s e d as c o u n t s / m i n p e r g t h y r o i d t i s s u e p e r 45-rain i n c u b a t i o n .
Substance
Amount per flask
14C02 (counts/min per g)
Control
--
13 442 ± 287
Vasopressin
2
TSH
o.25 u n i t s
Vasopressin, T S H
2
units
12 781 :~ 892 31 704 ~ 1812
units, 0.25 u n i t s 21 791 :~ 469
DISCUSSION
The observation that T S H increased T P N levels in thyroid slices and caused an equivalent decrease in DPN levels suggested an effect of the hormone on the enzyme DPN kinase which converts ATP and DPN to T P N (refs. I, 2). Since the addition of TSH to thyroid homogenates had no effect on DPN kinase activity, this enzyme was measured in homogenates prepared from slices incubated with and without TSH. Although the data in Table I I indicate that homogenates obtained from TSH-treated slices and incubated with ATP and DPN contained more T P N than did homogenates prepared from control slices, it does not necessarily signify augmented D P N kinase activity since net T P N synthesis was not obtained. The addition of A T P and D P N was associated with higher levels of T P N in the homogenate prepared from the TSH exposed slices, but they were still consistently less than the amount present at the end of the slice incubation. The results obtained could be equally well explained by an inhibition of destruction of T P N in the presence of ATP and D P N by the homogenate prepared from slices incubated with TSH. Since these experiments did not discriminate between the possibility of increased T P N synthesis and inhibition of T P N degradation, studies were done in which "~PN formation was measured in terms of [I-14CJ-6-phosphogluconate oxidation to 14CO~. In such a system T P N might be utilized for oxidation of [I-14C]-6-phosphogluconate before being destroyed. In this system the evolution of 14CO2 was not limited by 6-phosphogluconic dehydrogenase and was related to the amount of T P N present (unpublished observations). To correct for the initially higher T P N in the homogenate obtained from T S H incubated slices homogenates were kept at 37 ° for Biochim. Biophys. Acta, 121 (1966) 241-249
24S
j.B. WELD et al.
I h in the absence of nicotinamide. At the end of this time endogenous TPN was very low in both homogenates (Table III). Addition of various amounts of ATP and DPN increased the oxidation of [I-~4C~-6-phosphogluconate and at any level of ATP and DPN the increment was greater using homogenate obtained from the TSH incubated slices. These results are consistent with net TPN synthesis and increased DPN kinase activity in homogenate prepared from slices incubated with TSH. The data indicate that the concentration of DPN influenced 6-phosphogluconate oxidation more than that of ATP. This observation is in contrast to our previous finding that moderate reduction of DPN in thyroid slices by incubation with Carzinophilin did not inhibit the T S H effect on either glucose oxidation or increased TPN synthesis 4. t;urthermore the importance of ATP in the T S H effect on thyroid slices is suggested by the inhibition of TSH stimulation by metabolic inhibitors which presumably act by impairing ATP generation 14. One possible explanation for this apparent discrepancy between slice and homogenate results is that endogenous ATP in homogenates is present in excess and the rate of destruction of DPN is accelerated to the point where it becomes rate limiting. Although these experiments demonstrate an effect of TSH on DPN kinase activity, they do not elucidate the importance of intracellular concentrations of ATP and DPN in respect to TPN synthesis in intact thyroid cells. Most of the DPN kinase activity was localized in the nuclear fraction contrary to the results of CLAI~Kel al. '~ who reported the activity in the supernatant of liver cells. The TSH-mediated increase in TPN in thyroid slices was found in the supernatant (Table I). Such localization could represent leakage of TPN into the supernatant from another subcellular component during fractionation of the cells. The nuclear localization of DPN kinase would support this possibility. Since previous results had demonstrated that the TSH stimulation was independent of new protein synthesis°- it was not suprising that actinomycin D which inhibits RNA synthesis t6 did not modify the augmented glucose oxidation of thyroid slices exposed to T S H (Table VI). The concentration used was sufficient to inhibit ~8J~C]adenine incorporation into nucleic acids by 9 ° °o yet it did not influence basal or hormonal stimulation of glucose oxidation. Although it is currently fashionable to implicate RNA synthesis in hormone action '7 it is apparent that effects of TSH on glucose oxidation and TPN synthesis can not be explained by such a mechanism. The thyroid gland shares with gastric mucosa and salivary gland the ability to concentrate iodide. However, the failure of TSH to influence glucose oxidation in gastric mucosa and submandibular gland is consistent with its inability to stimulate iodide concentration in these tissues. Indirect evidence has been presented suggesting a direct stimulatorv effect of vasopressin on thyroid gland function12,13. If vasopressin does mimic TSH in stimulating thyroxine release it does not do so as a result of increasing glucose oxidation (Table VIII). An amount of vasopressin which by itself had no effect on basal glucose oxidation actually partially inhibited the action of TSH. The mechanism of such inhibition is not evident at present. ACKNOWLEDGEMENT
This investigation was supported by U.S. Public Health Service Grant AMo6865 from the National Institutes of Health. Biochim. Biophys. Acta, 121 (I966) 241- 249
TSH
EFFECTS ON THYROID D P N
KINASE
249
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Biochim. Biophys. Acta, 121 (1966) 241-249