뇌하수체척출(剔出)이 정상 및 cholesterol 투여 백서의 각장기내 지질분포에 미치는 영향에 관한 형태학적 관찰

Abstract

[한글]

A Histological Study on the Effects of Hypophysectomy in Normal and Cholesterol-fed

Rats; With Special Reference to Tissue Lipid Distribution

In Joon Choi, M.D.

Department of Pathology, College of Medicine, Yonsei University Seoul, Korea

(Directed by Prof. Dong Sik Kim, M.D.)

The relation between plasma lipids and atherosclerosis has been suspected for a

long time and the importance of lipid metabolism in the pathogenesis of

atherosclerosis has since been recognized. In view of the association between high

levels of plasma lipids and atherosclerosis, there have been exceedingly numerous

inverstigations to examine the factors involved in such influence.

Among the factors related to disturbances in lipid metabolism, study on the role

of the endocrine glands and of various hormones has attracted many workers in this

field to clarify the mechanisms involved in such phenomena.

Investigation of the literatures reveals that the pituitary, adrenal glands and

gonads all plays a significant role in the regulation of lipid metabolism. Seifter

et al. and Zarafonetis et al. isolated the so-called "lipid-mobilizing hormone" in

the plasma, and found it to he more abundant in the posterior lobe of the pituitary

than the anterior lobe. Chalmers et al. extracted a "fat-mobilizing substance" in

the urine which was eliminated after hypophysectomy. Rudman et al. also

demonstrated a similar substance in an alkaline extraction of the anterior

pituitary and named it Fraction H. More recently, Williams reported that these

substance must be the growth hormone and ACTH.

The association of hypercholesterolemia and hypothyroidism is a well known

clinical phenomenon and also this has been proven experimentally (Hurxthal et al.,

Wells and Ershoff). There are some who believe that the alterations of lipid

metabolism and the development of atherosclerosis following hypophysectomy were due

to the resultant hypothyroidism and not as a direct effect of hypopituitarism.

The intimate relationship of pituitary and adrenal glands is also very well

known. The role of sex hormone, particularly of estrogen, attracted many

investigators to examine the various factors related to the regulation of lipid

metabolism. Thus, it is quite apparent that the endocrine glands are very

intimately associated with the alterations of lipid metabolism and that the

pituitary gland plays the central role.

However, the majority of these studies have dealt mainly with the fluctuation of

plasma lipid levels and very little attention has bean paid to the lipid

distribution in the various parenchymatous organs, particularly of the endocrine

glands. It is also recognized that the level of plasma lipid alone cannot

thoroughly explain the variegated features of diseases resulting from disturbances

in lipid metabolism. Therefore, study of tile pattern of tissue lipid distribution

in normal and abnormal conditions seems to be quite appropriate and important.

Furthermore, there are still wide differences of opinions as to the mode of action

of the various endocrine factors in the regulation of lipid metabolism.

The purpose of this study is to characterize the pattern of lipid distribution in

various organs after hypophysectomy in normal and cholesterol-fed rats. At the same

time, the influence of cortisone, estrogen, testosterone and thyroxine was

evaluated as to the mechamism(s) involved in their action toward tissue lipid

alterations.

Materials and Methods:

Healthy albino rats of both sexes weighing from 100 to 250 grams were used.

Although a total of 166 rats were subjected to hypophysectormy, the number of

hypophysectomized rats included in the final study was 32 (male 21, female 11)

which survived successful total excision of pituitary. The animals were divided

into 3 major groups: GroupⅠ consisted of 6 rats as normal control group: GroupⅡ

consisted of 12 rats as the cholesterol only feeding group; GroupⅢ consisted of 32

rats which had hypophysectomy. The hypophysectomy group was subdivided into six

groups as follows: Group A consisted of 7 rats haying hypophysectomy only; Group B

consisted of 10 rats receiving cholesterol: Group C consisted of 4 rats receiving

thyroxine plus cholesterol; Group D consisted of 5 rats receiving cortisone plus

cholesterol; GroupE consisted of 3 rats receiving estrogen plus cholesterol: Group

F consisted of 3 rats receiving testosterone plus cholesterol.

Hypophysectomy was performed by the transauricular method. All animals after

hypophysectomy received 0.01 mg./gm. body weight of cortisone, 0.05cc/gm of 5%

glucose solution into the peritoneal cavity, and 0.05mg/gm. body weight of

sigmamycin intramuscularly for 2 successive days. Each extracted pituitary was

examined under the microscope every time to confirm its total excision. This was

checked again at the time of autopsy and the partially removed cases were excluded

from the final analysis.

300 mg. of cholesterol was given daily starting two days after hypophysectomy

until the end of the experiment. The dose of thyroxine was 0.5 mg./kg. per day,

cortisone 0.5 mg/kg. per day, estrogen 0.02 mg/kg. per every other day as was

testosterone, 0.42mg/kg. These were given for the last 20 days of the experiment.

The animals were sacrificed at intervals of 3, 5 and 8 weeks. The organs were

grossly inspected and fixed in 10% neutral formalin. The fresh weight of the

thyroid, adrenals, testes and ovaries was recorded Paraffin sections were prepared

for hematoxylin and rosin, and for Periodic Acid Schiff's reaction. Frozen sections

were stained with Oil Red 0 for the demonstration of fat in the tissues.

Results:

1. Normal control group (Group Ⅰ); The adrenal glands showed intense

sudanophilic lipid droplets in all layers but their intensity was more marked in

the zona glomerulosa. The bona fasciculata contained slightly less and the bona

reticularis the least. There was a very narrow lipid-free zone between the zona

glomerulosa and zona fasciculata.

All of the other organs examined showed the usual histological appearance. Fat

stanis of the thyroid, liver and kidney failed to reveal stainable fat, but fat

stains of the testes and ovary showed the usual lipid distribution.

2. Cholesterol only feeding group (GroupⅡ); Cholesterol feeding in normal rats

caused considerable changes in the various organs. The adrenal glands not only

showed increase in weight and size but also the lipid drop키eta were markedly

increased both in number and size. All layers contained prominent lipid droplets.

At the end of 8 weeks of cholesterol feeding, the lipid-free zone in the normal

adrenals became also filled with lipid. The liver also demonstrated fine

sudanophilic droplets within the Kupffer cells and the hepatic cells at the end of

3 weeks which became progressively intensified as the time lapsed. The kidney

showed demonstrable lipid droplets in the glomeruli and in the distal tubules.

The thyroid showed features of follicular hyperplasia but no significant lipid

droplets. In many areas the lining epithelium skewed papillary infoldings and the

cytoplasm contained many PAS positive materials. The testes and ovary were not

significantly altered when compared with the normal control group.

3. Hypophysectomy group (GroupⅢ); As expected the hypophysectomy produced

profound morphologic alterations. The adrenal glands showed atrolhy, but the

relative size of the zona glomerulosa, particularly of the subglomerulosal layer

(lipid-free zone), was progressively broadened as the time lapsed. The zone

fasciculata was extremely atrophic, on the other hand, and the zona reticularis was

also thinned. The examination of lipid pattern revealed that the zone glomerulosa

persistently showed demonstrable lipids throughout the experiment but the other

zones demonstrated progressive loss of lipid substance.

In this group the lipid droplets in the liver and kidney became demonstrable

which was not the case in normal control animals. The thyroid showed marked

flattening of the follicular lining epithelium, storage of colloid substance, and

no significantly demonstrable lipids. The testes and ovaries showed varying degrees

of atrophy, in association with reduced lipid pattern in the ovary, but there was a

significantly increased irregular and larger lipid materials in the seminiferous

tubules of the testes.

When cholesterol was fed to this group of animals, there were no significant

histological difference from the group having hypophysectomy only but the lipid

content of the adrenal glands was greater. However this increase was less than that

seen in the normal control animals. The liver and the kidney both revealed that

there were increase in lipid comparable to that seen in cholesterol-fed animals

without hypophysectomy. The thyroid, testes and ovary all showed changes similar to

the group having hypophysectomy only.

Administration of thyroxine did not affect the lipid distribution of adrenal

cortex, thyroid, ovary, and testes but the lipid un hepatic lobules and kidney

tubules were markedly reduced.

Cortisone administration caused lipid depletion in the adrenal glands but the

thyroid, liver and kidney showed no significant alteration of lipid pattern.

Estrogen and testosterone likewise did not influence the pattern of lipids in the

liver and kidney. However estrogen increased the adrenal and ovarian lipids and

testosterone, the adrenal and testicular lipids.

Conclusion:

1. Cholesterol feeding caused significant alterations in the tissue lipid

distrioution, particularly of the adrenal glands, liver and kidney. The thyroid

gland became hyperplastic.

2. Hypophysectomy alone induced atrophic changes of the examined endocrine organs

and alterations in the lipid distribution of the adrenals, liver, kidney, ovary and

testes.

3. Cholesterol supplementation in hypophysectomized animals caused no significant

alterations in the pattern of tissue lipid distribution as compared with that of

the cholesterol-fed rats, but the intensity and the amount of lipid was much less

in the former.

4. Administration of thyroxine caused profound influences on the lipids seen in

the liver and kidney but none on the adrenal lipids. On the other hand, cortisone,

estrogen and testosterone influenced lipid pattern of the adrenal glands but not of

the liver and kidney. It appears that the thyroid gland influences the lipid

distribution of the adrenal glands through the mediation of pituitary gland, but

is directly concerned with the lipid pattern of the liver and kidney. Cortisone

does not appear to act directly on the lipid pattern of the liver and kidney

although it is directly related to the adrenal lipids. Estrogen and testosterone

also do not appear to directly influence the pattern of the liver and kidney lipid

but estrogen may be directly related to the alterations of the lipid in the inner

cortex of the adrenals and of the ovary. Also testosterone may be related directly

to the alterations of the lipid in the inner cortex of the adrenals and of the

testes.

It appears that alterations in the tissue lipid pattern of the liver and kidney

may be related to the resultant hypothyroidism following hypophysectomy. On the

contrary the lipid pattern of the adrenal, ovary and testes may be related directly

to the effect of the hypophysectomy.

[영문]

The relation between plasma lipids and atherosclerosis has been suspected for a long time and the importance of lipid metabolism in the pathogenesis of atherosclerosis has since been recognized. In view of the association between high levels of plasma lipids and atherosclerosis, there have been exceedingly numerous

inverstigations to examine the factors involved in such influence.

Among the factors related to disturbances in lipid metabolism, study on the role of the endocrine glands and of various hormones has attracted many workers in this field to clarify the mechanisms involved in such phenomena.

Investigation of the literatures reveals that the pituitary, adrenal glands and gonads all plays a significant role in the regulation of lipid metabolism. Seifter et al. and Zarafonetis et al. isolated the so-called "lipid-mobilizing hormone" in the plasma, and found it to he more abundant in the posterior lobe of the pituitary than the anterior lobe. Chalmers et al. extracted a "fat-mobilizing substance" in the urine which was eliminated after hypophysectomy. Rudman et al. also demonstrated a similar substance in an alkaline extraction of the anterior pituitary and named it Fraction H. More recently, Williams reported that these

substance must be the growth hormone and ACTH.

The association of hypercholesterolemia and hypothyroidism is a well known clinical phenomenon and also this has been proven experimentally (Hurxthal et al., Wells and Ershoff). There are some who believe that the alterations of lipid metabolism and the development of atherosclerosis following hypophysectomy were due

to the resultant hypothyroidism and not as a direct effect of hypopituitarism.

The intimate relationship of pituitary and adrenal glands is also very well known. The role of sex hormone, particularly of estrogen, attracted many investigators to examine the various factors related to the regulation of lipid metabolism. Thus, it is quite apparent that the endocrine glands are very intimately associated with the alterations of lipid metabolism and that the

pituitary gland plays the central role.

However, the majority of these studies have dealt mainly with the fluctuation of plasma lipid levels and very little attention has bean paid to the lipid distribution in the various parenchymatous organs, particularly of the endocrine glands. It is also recognized that the level of plasma lipid alone cannot

thoroughly explain the variegated features of diseases resulting from disturbances in lipid metabolism. Therefore, study of tile pattern of tissue lipid distribution in normal and abnormal conditions seems to be quite appropriate and important.

Furthermore, there are still wide differences of opinions as to the mode of action of the various endocrine factors in the regulation of lipid metabolism.

The purpose of this study is to characterize the pattern of lipid distribution in various organs after hypophysectomy in normal and cholesterol-fed rats. At the same time, the influence of cortisone, estrogen, testosterone and thyroxine was evaluated as to the mechamism(s) involved in their action toward tissue lipid alterations.

Materials and Methods:

Healthy albino rats of both sexes weighing from 100 to 250 grams were used.

Although a total of 166 rats were subjected to hypophysectormy, the number of hypophysectomized rats included in the final study was 32 (male 21, female 11) which survived successful total excision of pituitary. The animals were divided into 3 major groups: GroupⅠ consisted of 6 rats as normal control group: GroupⅡ consisted of 12 rats as the cholesterol only feeding group; GroupⅢ consisted of 32 rats which had hypophysectomy. The hypophysectomy group was subdivided into six groups as follows: Group A consisted of 7 rats haying hypophysectomy only; Group B

consisted of 10 rats receiving cholesterol: Group C consisted of 4 rats receiving thyroxine plus cholesterol; Group D consisted of 5 rats receiving cortisone plus cholesterol; GroupE consisted of 3 rats receiving estrogen plus cholesterol: Group F consisted of 3 rats receiving testosterone plus cholesterol.

Hypophysectomy was performed by the transauricular method. All animals after hypophysectomy received 0.01 mg./gm. body weight of cortisone, 0.05cc/gm of 5% glucose solution into the peritoneal cavity, and 0.05mg/gm. body weight of sigmamycin intramuscularly for 2 successive days. Each extracted pituitary was examined under the microscope every time to confirm its total excision. This was checked again at the time of autopsy and the partially removed cases were excluded from the final analysis.

300 mg. of cholesterol was given daily starting two days after hypophysectomy until the end of the experiment. The dose of thyroxine was 0.5 mg./kg. per day, cortisone 0.5 mg/kg. per day, estrogen 0.02 mg/kg. per every other day as was testosterone, 0.42mg/kg. These were given for the last 20 days of the experiment.

The animals were sacrificed at intervals of 3, 5 and 8 weeks. The organs were grossly inspected and fixed in 10% neutral formalin. The fresh weight of the thyroid, adrenals, testes and ovaries was recorded Paraffin sections were prepared for hematoxylin and rosin, and for Periodic Acid Schiff's reaction. Frozen sections were stained with Oil Red 0 for the demonstration of fat in the tissues.

Results:

1. Normal control group (Group Ⅰ); The adrenal glands showed intense sudanophilic lipid droplets in all layers but their intensity was more marked in the zona glomerulosa. The bona fasciculata contained slightly less and the bona reticularis the least. There was a very narrow lipid-free zone between the zona

glomerulosa and zona fasciculata.

All of the other organs examined showed the usual histological appearance. Fat stanis of the thyroid, liver and kidney failed to reveal stainable fat, but fat stains of the testes and ovary showed the usual lipid distribution.

2. Cholesterol only feeding group (GroupⅡ); Cholesterol feeding in normal rats caused considerable changes in the various organs. The adrenal glands not only showed increase in weight and size but also the lipid drop키eta were markedly increased both in number and size. All layers contained prominent lipid droplets.

At the end of 8 weeks of cholesterol feeding, the lipid-free zone in the normal adrenals became also filled with lipid. The liver also demonstrated fine sudanophilic droplets within the Kupffer cells and the hepatic cells at the end of 3 weeks which became progressively intensified as the time lapsed. The kidney showed demonstrable lipid droplets in the glomeruli and in the distal tubules.

The thyroid showed features of follicular hyperplasia but no significant lipid droplets. In many areas the lining epithelium skewed papillary infoldings and the cytoplasm contained many PAS positive materials. The testes and ovary were not significantly altered when compared with the normal control group.

3. Hypophysectomy group (GroupⅢ); As expected the hypophysectomy produced profound morphologic alterations. The adrenal glands showed atrolhy, but the relative size of the zona glomerulosa, particularly of the subglomerulosal layer (lipid-free zone), was progressively broadened as the time lapsed. The zone fasciculata was extremely atrophic, on the other hand, and the zona reticularis was also thinned. The examination of lipid pattern revealed that the zone glomerulosa persistently showed demonstrable lipids throughout the experiment but the other

zones demonstrated progressive loss of lipid substance.

In this group the lipid droplets in the liver and kidney became demonstrable which was not the case in normal control animals. The thyroid showed marked flattening of the follicular lining epithelium, storage of colloid substance, and no significantly demonstrable lipids. The testes and ovaries showed varying degrees of atrophy, in association with reduced lipid pattern in the ovary, but there was a significantly increased irregular and larger lipid materials in the seminiferous tubules of the testes.

When cholesterol was fed to this group of animals, there were no significant histological difference from the group having hypophysectomy only but the lipid content of the adrenal glands was greater. However this increase was less than that seen in the normal control animals. The liver and the kidney both revealed that there were increase in lipid comparable to that seen in cholesterol-fed animals without hypophysectomy. The thyroid, testes and ovary all showed changes similar to the group having hypophysectomy only.

Administration of thyroxine did not affect the lipid distribution of adrenal cortex, thyroid, ovary, and testes but the lipid un hepatic lobules and kidney tubules were markedly reduced.

Cortisone administration caused lipid depletion in the adrenal glands but the thyroid, liver and kidney showed no significant alteration of lipid pattern.

Estrogen and testosterone likewise did not influence the pattern of lipids in the liver and kidney. However estrogen increased the adrenal and ovarian lipids and testosterone, the adrenal and testicular lipids.

Conclusion:

1. Cholesterol feeding caused significant alterations in the tissue lipid distrioution, particularly of the adrenal glands, liver and kidney. The thyroid gland became hyperplastic.

2. Hypophysectomy alone induced atrophic changes of the examined endocrine organs and alterations in the lipid distribution of the adrenals, liver, kidney, ovary and

testes.

3. Cholesterol supplementation in hypophysectomized animals caused no significant alterations in the pattern of tissue lipid distribution as compared with that of the cholesterol-fed rats, but the intensity and the amount of lipid was much less in the former.

4. Administration of thyroxine caused profound influences on the lipids seen in the liver and kidney but none on the adrenal lipids. On the other hand, cortisone, estrogen and testosterone influenced lipid pattern of the adrenal glands but not of the liver and kidney. It appears that the thyroid gland influences the lipid distribution of the adrenal glands through the mediation of pituitary gland, but is directly concerned with the lipid pattern of the liver and kidney. Cortisone does not appear to act directly on the lipid pattern of the liver and kidney

although it is directly related to the adrenal lipids. Estrogen and testosterone also do not appear to directly influence the pattern of the liver and kidney lipid but estrogen may be directly related to the alterations of the lipid in the inner cortex of the adrenals and of the ovary. Also testosterone may be related directly to the alterations of the lipid in the inner cortex of the adrenals and of the testes.

It appears that alterations in the tissue lipid pattern of the liver and kidney may be related to the resultant hypothyroidism following hypophysectomy. On the contrary the lipid pattern of the adrenal, ovary and testes may be related directly to the effect of the hypophysectomy.