fa)

Vol. 4, pp. 589-591. Printed in the U.S..4

Brain Research Bdktin,

Elevation of Calcitonin Immunoreactivity in the Pituitary and Thyroid Glands of Genetically Obese Rats (falfa)’ DAVID L. MARGULES, Department

JAMES J. FLYNN,

JOSEPH

WALKER

AND CARY W. COOPER2

Department of Psychology, Temple University, Philadelphia, PA 19122 and of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27514

Received

3 July 1979

MARGULES, D. L., J. J. FLYNN, J. WALKER AND C. W. COOPER.

Elevation

oj’calcitonin

immrtnoreactit?ty

in ~/re

(fa/fa). BRAIN RES. BULL. 4(5) 589-591, 1979.-This paper contains the first demonstration of quantitative changes in the levels of calcitonin-like immunoreactivity in the pituitary. The concentration of calcitonin-like material in lean Zucker rats (?I+) was 0.42 + 0.09 ng/mg wet weight of pituitary. The pituitaries of obese rats (fafa) contained significantly greater levels (1.56 t 0.56 ngimg wet weight). This 271% increase represents the first indication that pituitary calcitonin-like material may have a physiological role in genetically obese rats. Thyroidal calcitonin also was elevated in the obese (111%). These large elevations in the calcitonin content of the glands of obese rats were not accompanied by significant elevations of calcitonin in the blood. This suggests that obese rats have problems with mechanisms for the release of calcitonin into the blood and might not be able to combat hypercalcemia as effectively as leans. However, this hypothesis remains to be tested.

pituitary

Calcitonin

and

thyroid

glands

of generically

lmmunoreactivity

obese

rats

Pituitary gland

Thyroid gland

CALCITONIN originates from C-cells in the thyroid gland of mammals and acts to lower blood levels of calcium by inhibiting the movement of calcium from the skeleton into the bloodstream [ 11. Recently, calcitonin-like immunoreactive material has been reported to occur in all cells of the intermediate lobe and scattered cells of the anterior lobe of the rat pituitary gland [2,3]. This conclusion was based on immunochemical staining of pituitaries [2,3] and on finding immunological activity in ovine and porcine pituitary extracts [2]. This finding has surprised many investigators. Calcitonin has never before been reported in the pituitary and it is not thought of as a pituitary hormone. Initially, Deftos and coworkers [2] were not able to detect calcitonin in the rat pituitary by radioimmunoassay (RIA). Thus, their immunofluorescent study opened up many questions about the status of calcitonin. In this paper we report the first quantitative (RIA) evidence of baseline levels of immunoreactive calcitonin-like material in the rat pituitary. These were established in lean adult rats. Also, we report for the first time an elevated level of calcitonin in pituitary and thyroid glands of genetically obese rats. These findings confirm and extend the work of Deftos and coworkers [2,3] and open up new possibilities for a physiological role of calcitonin, particularly in connection with the control of obesity.

Obesity

METHOD

Eighteen Zucker rats were purchased from Bird Laboratories at the age of two months. They were maintained in our laboratory for eight months before this experiment began. Eight of these were genetically obese (fulfl) and ten were lean controls (?/+). The obese rats consisted of five females and three males, and the lean rats of six females and four males. There were no significant sex-related differences in any of our assays or measures with the exception of the well-known tendency for males to have a greater body weight than females. Therefore, data from the sexes were combined. All animals were fed Purina laboratory chow (No. 5001) and water ad lib. Half of the rats had access to a milk dessert in addition to their chow. This consisted of 1 hr of access to sucrose-sweetened reconstituted whole milk at 12.00 hr. There were no significant milk-related differences in any of our measures or assays and data from rats with and without histories of milk access were combined. The animals were kept in individual hanging cages made of wire mesh and had 12 hr of light alternating with 12 hr of darkness. Light onset occurred at 6.00 and offset at 18.00 hr. Room temperature varied between 21” and 24°C. Animals were sacrificed between 12.00 and 13.00 hr by quick decapitation. No animal had access to milk on the day of sacrifice. Trunk blood was collected and chilled in poly-

‘Supported by Grant BNS-77-22630 from the National Science Foundation and U.S.P.H.S. Grant AM-17743 from the National Institute of Arthritis, Metabolism and Digestive Diseases. ‘We thank Mr. Johnny F. Obie and Ms. Deloris B. Alston for expert technical assistance.

CoDvrkht

o 1979 ANKHO International

Inc.-0361-9230/79/050589-03$00.80/O

MARGULES c; of

I ‘PO

I ma

lean (1

I 3?0

ET AL.

*I I 4?0

I

I

5Qf

St0

w 366

1

720

1010

body wgt.

k 6.2

12.4

18.6

21:a

ng CT pituitary

pwntary

6661

9334

3i.5

6f

1.26

1.68

la601

18661

gland

wgt. Imgl

ng CT mg pituitary

“g CTf;thymid

gland

thyroid wgf. (mg)

323

493.5

&I

2k

3;

tk

id0

Go

I

I

I

I

I

I

ng CTimg thyroid

F 1619

mlmdkihr

FIG. I. Values for various measures in fat and lean Zucker rats are compared. Scales for both absolute values and percent of lean controls are shown. White bars represent the mean values for lean rats and black bars for fat rats. Brackets represent plus one standard error of the mean.

propylene tubes. Pituitaries and thyroids were dissected under microscopic guidance, frozen on dry ice within 5 min of sacrifice. and stored at -48°C until assay. Serum was separated from the clot by low speed centrifugatjon at 4°C and then was stored at -48°C. Immunoreactive calcitonin and caicitonin-bike material were measured using RIA procedures described previously in detail [4,5]. To analyze pituitaries, the antiserum used was one raised against synthetic human calcitonin which crossreacts with rat calcitonin. This antibody stains rat pituitary cells (immunoperoxidase procedure) exactly in the manner reported by others [2.3]. For RIA, the antiserum was employed at a final dilution of I: 150.000. Pure, synthetic human calcitonin was used for labeling with 9, and pure, native rat calcitonin was used as the unlabeled reference standard. Separation of bound and free hormone was accomplished by the second antibody procedure. To analyze thyroids and serum. the antiserum used was one raised against native rat calcitonin. It was used at a final

dilution of 1: 10,000. and pure, native rat calcitonin was used both for labeling with IpZI and as the unlabelled reference standard [4.5]. For RIA, pituitary glands were homogenized individually in 0.5 ml lO--:‘N HCI in 0.15 y NaCi at 4°C and tested at multiple dilutions (highest concent~tion tested = 100 i(~1per 500 ~1 incubation vob. Thyroid glands were homogenized individually in a 2 ml vol of HCI-NaCI and tested at multiple dilutions (highest concentration = 1.25 ~1 per 500 J.J incubation vol.). Sera were assayed directly as described previously [Sl. For both pituitary and thyroid extracts, the immunoreactive material produced dose-dependent effects in the RIA which were indistinguishable from the calcitonin standards. Data were analyzed by unpaired two-tailed r-tests. RESULTS

Our results are summarized

in Fig. I _The obese rats had a

CALCITONIN

IN OBESE RATS

591

significantly greater (p
This is the first quantitative

report

of levels

munoreactive calcitonin-like material in rat pituitaries. In the recent reports by Deftos er al. [2,3], calcitonin was localized in cells of the intermediate lobe and in scattered cells in the anterior lobe. These authors were unable to quantify rat pituitary calcitonin levels by radioimmunoassay. We cannot easily explain their failure and our success. Their lean rats differed from ours in both strain and age; both variables have been shown to affect thyroid calcitonin levels [6,7]. The exact nature of the pituitary calcitonin-like material remains to be clarified. However, Nakanishi et al. [8] have recently predicted the peptide sequence of the 3 1,000 dalton precursor peptide for bovine ACTH-P-LPH. In the cryptic portion of the peptide, N-terminal to ACTH, there is a 32 amino acid sequence showing -25% homology with calcitonin. Therefore, it seems reasonable that this region of homology may explain the immunoreactivity we and others have observed in the pituitary. The function of this peptide, if any, remains to be determined. Comparison of the calcitonin levels of our obese rats to those of the lean Fischer 344 [7] may help to explain our results. Both strains have elevated calcitonin contents in their thyroids (461 2 38 for the Fischer and 344 ? 67 ng/mg for thefuifLl rats). The Fischer 344 is one of the few strains that remains lean throughout its lifespan. This strain not only has elevated calcitonin in its thyroid, it also can release calcitonin into the blood as indicated by its basal serum level of 1.52 + 0.06 r&ml [7]. The obese rats apparently lack this capacity and have instead a highly sporadic pattern (10.1 * 6.7 ngiml) that does not differ significantly from zero. Recent evidence exists suggesting that calcitonin has powerful and long lasting suppressant effects on feeding [9]. Thus, the inability to release calcitonin into the blood in a consistent pattern could contribute to the overeating problem of thefrrifrr rat.

of imREFERENCES

Munson, P. L. Physiology and pharmacology of thyrocalcitonin. In: Handbook qf Phsioloyy, Vol. VII, edited by G. D. Aurbach. Washington, DC: Am. Physiol. Sot., 1976, pp: 443-464. Deftos. L. J.. D. Burton. H. B. Bone. B. D. Catherwood. J. G. Parthemore. ‘R. Y. Moore, S. Minick and R. Guillemin. Immunoreactive calcitonin in the intermediate lobe of the pituitary gland. Life Sri. 23: 743-748, 1978. Deftos, L. J., D. Burton, B. D. Catherwood, H. G. Bone, J. G. Parthemore, R. Guillemin, W. Watkins and R. Y. Moore. Demonstration by immunoperoxidase histochemistry of calcitonin in the anterior lobe of the rat pituitary. J. clin. Endocr. Metab. 47: 457-459, 1978. But-ford, H. J., D. A. Ontjes, C. W. Cooper, A. F. Parlow and P. F. Hirsch. Purification, characterization and radioimmunoassay of thyrocalcitonin from rat thyroid glands. Endocrinology 96: 34&348,

197.5.

5. Cooper,

6.

C. W., J. F. Obie and W. H. Hsu. Improvement and initial in vivo application of the radioimmunoassay of rat thyrocalcitonin. Proc. Sot. e.rp. Eiol. Med. 151: 183-188, 1976. Cooper, C. W. Strain differences in thyrocalcitonin content of rat thyroid glands associated with differences in sensitivity to thyrocalcitonin. Endocrinology 82: 1015-1020, 1968. Peng, T.-C, C. W. Cooper and S. C. Garner. Thyroid and thyrocalcitonin concentrations and C-cell abundance in two strains of rats at different ages. Prw. Sot. rxp. Biol. Med. 153: 268-272.

1976.

Nakanishi, S., A. Inoue, T. Kita. M. Nakamura, A. C. Y. Chang, S. N. Cohen and S. Numa. Nucleotide sequence of cloned cDNA for bovine corticotropin-P-lipotropin precursor. Nrcmre 278: 423-427. 1979. Freed. W. J., M. J. Perlow. J. S. Carman and R. J. Wyatt. Calcitonin and feeding. Sot. Nenrosci. Absrr. 4: 428. 1978.