Metabolic and immunological responses associated with in vivo glucocorticoid depletion by...

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Life Sciences 73 (2003) 3159–3174

Metabolic and immunological responses associated with in vivo

glucocorticoid depletion by adrenalectomy in mature

Swiss albino rats

B. Bishayi*, S. Ghosh

Immunology Laboratory, Department of Physiology, University Colleges of Science and Technology, University of Calcutta,

92, A.P.C. Road, Kolkata-700009, West Bengal, India

Received 14 February 2003; accepted 11 June 2003

Abstract

The study is undertaken to determine the effect of adrenal corticosteriod depletion after adrenalectomy on

carbohydrate, protein and fat metabolism as well as maturation and functional efficacy of the

immunocompetent cells. Beside biochemical and hematological parameters, whether in vivo glucocorticoid

depletion has any modulatory effects on splenic macrophage responses to bacterial challenge with regards to

intracellular killing, nitric oxide release and cellular integrity, were determined. Major findings of our study

indicate that blood glucose, urea and total inorganic phosphate levels showed a time dependent increase in

adrenalectomized rats compared to control. Total glycogen content in liver was decreased gradually due to

adrenal corticosteroid insufficiency. Hematological parameters like hemoglobin concentration, hematocrit value,

total leukocyte count and differential count were also found to increase in the adrenalectomized group with

respect to intact group. From the functional study of immunocompetent cells, intracellular killing capacity of

splenic macrophages recovered from control and adrenalectomized rats after 10 and 20 days of adrenalectomy

showed no significant alteration; however, the function of splenic macrophages recovered from rats after 30

days of adrenalectomy showed altered response. Nitric oxide released from splenic macrophages of

adrenalectomized rats was less than that of control animal even after stimulation with lipopolysaccharide. DNA

fragmentation assay showed a lesser degree of fragmentation of splenic macrophages obtained from

adrenalectomized rats indicating, apoptotic death of cells in this group decreases. Adrenal corticosteroid

insufficiency due to adrenalectomy interferes with metabolic and hematopoietic functions and modulates the

0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.lfs.2003.06.009

* Corresponding author. Fax: +91-033-351-9755.

E-mail address: [email protected] (B. Bishayi).

B. Bishayi, S. Ghosh / Life Sciences 73 (2003) 3159–31743160

development and maintenance of normal immunitary status, which in turn influences the inflammatory

response.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Glucocorticoids; Adrenalectomy; Immunomodulation; Rat macrophage function

Introduction

Glucocorticoids (GC) synthesized in the cortex of adrenal gland are an essential component of the

stress-adaptive mechanism of the body and is a potent immuno-suppressive agent (Rook et al., 2000;

Long et al., 1940; Blaschko, 1975). GC also affects protein and carbohydrate metabolism and

modulate important metabolic activities (Casadevall et al., 1999). Loss of adrenal cortical functions

ultimately results in death unless hormone replacement therapy is instituted (Granner, 2000). The

pancreatic tissue of adrenalectomized rats secreted much less amount of insulin in response to

glucose administration not only in the early phase but also in the late phase of release pattern

(Kawai and Kuzuya, 1977). Generally insulin secretion is found to be depressed in adrenalectomized

animals in the post absorptive state and that increase in insulin secretion rate brought about by

hyperglycemia is less than normal (Debreceni et al., 1990). It has also been demonstrated that

pancreatic vagotomy decreases levels of plasma insulin in bilaterally adrenalectomized rats,

suggesting that adrenal glands have a masking action on vagal mechanism of insulin secretion

(Sakaguchi and Yamaguchi, 1980). Infusion of epinephrine in adrenalectomized rats is found to

restore stimulation of Glucose 6 phosphatase as triggered by hypoglycemia in normal rats (Bady et

al., 2002). Impaired hepatic glycogen synthesis in insulin dependent diabetic rats and in starved

adrenalectomized rats, have also been reported suggesting defective activation of glycogen synthase

(Kreutner and Goldberg, 1967). Glycogenin activity was reported to be similar in normal diabetic

and adrenalectomized fasted animals regardless of the hepatic glucose concentration (Gannon et al.,

1998). Deficiency in liver glycogen deposition following glucose administration in adrenalectomized

rats has long been appreciated (Britton and Silvette, 1932; Long et al., 1940). Bilateral adrenalec-

tomy also causes a disturbance of protein synthesis in peripheral tissues (Kudalaenko and Levshin,

1977; Stele, 1975). Corticosteroids are used to suppress pathological immune responses associated

with autoimmunity, inhibit rejection of allogenic tissues after organ transplantation and diminish

inflammation associated with a wide variety of hypersensitivity reaction (Blalock, 1989). Immuno-

modulatory effects of glucocorticoids have been demonstrated (Wilder, 1995; Dan and Lall, 1998).

GC exerts both positive and negative feedback regulatory mechanism towards gene expression of

various substances to combat inflammation (Murat and Barry, 2000). Depletion of endogenous GC

by adrenalectomy or by injection of saturating amounts of the GC receptor antagonist RU 38486

(mifepristone) leads to clonal deletion of peripheral T cells in vivo (Gonzalo et al., 1993).

Adrenalectomy also elevates prostaglandin production, cyclooxygenase enzyme synthesis and activity

in murine peritoneal macrophages (Vincent et al., 1986). Several studies have been performed to

investigate physiological as well psychological stress after unilateral or bilateral adrenalectomy on

single or combined parameters determining the functional activities of rats (Imura and Fukata, 1994).

However, few reports are available to identify the underlying process of the different metabolic

B. Bishayi, S. Ghosh / Life Sciences 73 (2003) 3159–3174 3161

activities in animals after adrenalectomy and to make use of adrenal corticosteroids for identification

of the mechanism involved.

To determine the importance of adrenal corticosteroids on metabolic activity we investigated changes

in several biochemical parameters in intact and unilaterally adrenalectomized (ADX) rats. Moreover,

whether in vivo glucocorticoid depletion leads to any modulatory role in the functions of immunocom-

petent cells after bacterial challenge with regards to intracellular killing was also determined. The study

may be helpful in understanding that adrenal corticosteroid insufficiency due to adrenalectomy interferes

with metabolic functions which may also support the hypothesis that endogenous GC plays a role in

maintaining metabolism.

Methods

Adrenalectomy

Adult male Swiss albino rats with average body weight 110 g were used for all experiments. Animals

were divided into control and unilaterally adrenalectomized group comprising of eight animals per

group. Unilateral adrenalectomy was done (to mimic a condition of adrenal hormone insufficiency) as

evident by surgical removal of the gland under ether anaesthesia with slight modification as described

earlier (Sakaguchi and Yamaguchi, 1980). Control animals were sham operated. Rats were housed in

plastic cages and kept under standard laboratory conditions with sufficient food and water and also with

proper avoidance of cruelty to animals wherever possible.

Collection of blood

Blood was collected from the retrorbital plexus of both control (sham operated) and unilaterally

adrenalectomized (ADX) rats on the 10th, 20th and 30th days after adrenalectomy. Serum was prepared

after clot retraction and kept at � 20 jC until further use.

Cortisol hormone assay

Serum cortisol level was directly measured by immuno-enzymatic assay method (Arakawa et al.,

1979) using the cortisol hormone assay kit. (Cortisol hormone assay kit for 48 Test from Equipar

diagnostics, Equipar srl, via G. Ferrari, 21/N-21047, Saronno (Va) Italy, in an ELISA-Strip Read-

er(Merck: MIOS-Mini).

Estimation of biochemical and hematological parameters

Blood glucose, total inorganic phosphate and serum concentration of urea were estimated from both

control and ADX rats as described earlier (Oser, 1965). Total protein in serum of control and ADX rats

were estimated by Lowry’s method (Lowry et al., 1951). Glycogen content of liver was determined by

Nelson-Somogyi method as described elsewhere (Plummer, 1999). Hematological parameters, like

concentration of Hemoglobin (Hb), Total leukocyte count (TC), Differential count (DC) and Hematocrit

value (Hct) were estimated as described elsewhere (Mukherjee, 1990).

Separation of splenic macrophages

Spleens were excised from sacrificed rat (both control and ADX groups) and immediately placed in

Alsever’s solution. They were then macerated separately using frosted glass slides. Cells were repeatedly

aspirated with a sterile Pasteur pipette until a single cell suspension was obtained. Suspension was then

transferred to sterile tubes and kept in ice for cell debris to settle. The supernatant was then layered over

3 ml Histopaque (1077) Sigma USA and then centrifuged at 1500 rpm for 30 min (Jessop et al., 1994).

After centrifugation, the band of leukocyte enriched fraction at the interface was collected and washed

with (Dulbecco’s phosphate buffered saline) DPBS; then the cell pellet was resuspended in RPMI-1640,

containing 20 mM hydroxy ethyl piperazine ethane sulfonic acid (HEPES), pH 7.2, 1 mg/ml Bovine

Serum Albumin (BSA) and was allowed to adhere on plastic surface for 1 hr in a 37 jC incubator. The

non-adherent cells were removed and adherent cells were collected by repeated aspiration with Pasteur

pipette. Cells were then washed and finally resuspended in culture media (RPMI + BSA) at a density of

106/ml. More than 95% cells were found viable as determined by Trypan blue dye exclusion technique.

Purity of the cells was checked by Giemsa Staining.

Preparation of bacteria (Staphylococcus aureus-MC 524) for phagocytosis and intracellular killing

To obtain bacteria in the mid-logarithmic phase, 50–200 Al of an overnight culture made in nutrient

broth was added in 50 ml of nutrient broth and incubated for 2.5 hr with orbital shaking. The cells were

washed in 10 mM sodium phosphate buffer (pH 7.4) and their concentration estimated by spectropho-

tometry at A620 on the basis of the relationship (Yao et al., 1997).

A6200:2 ¼ 5� 107cells=ml

Intracellular killing

Splenic macrophages recovered from both control and adrenalectomized rats were taken. Equal

volume of 1 � 107 macrophages/ml and bacteria in Hank’s balanced salt saline (HBSS)-gelatin were

incubated at 37 jC under slow rotation for 3 min. Phagocytosis was stopped by shaking the tubes in

crushed ice and free bacteria were removed by centrifugation and washing. Then cells containing

ingested bacteria were re-incubated at 37 jC for different times. At different time intervals cells were

taken and intracellular killing was terminated by keeping the cells in ice, followed by centrifugation.

After addition of distilled water containing 0.01% BSA to the pellet, cells were disrupted by vigorously

vortexing which causes release of intracellular bacteria in the supernatant. Then the viable intracellular

bacteria in the supernatant were determined microbiologically by colony counting method. The number

of colonies obtained at the beginning of the experiment (zero minute after reincubation) was designated

as given by 100% viable bacteria. Intracellular killing was then expressed as the percentage decrease in

the initial number of viable bacteria (Leigh et al., 1986).

Nitrix oxide (NO) release assay

Spleen cells (200 Al) were suspended in DPBS-BSA-glucose and were stimulated with 100 ng/ml

lipopolysaccharide (LPS) for 1 hr at 37 jC. After centrifugation at 10,000 rpm for 15 min, cell-free



supernatant were collected in a separate micro-centrifuge tube and was used for nitric oxide release

assay. Each of the cell free supernatant (200 Al) was reacted with Griess reagent (200 Al)containing 1 part of 1% sulfanilamide in 5% phosphoric acid and 1 part of 0.1% N-C-1 naphthyl-

ethylene-diamine-dihydrochloride was added and incubated for 10 min at room temperature.

Readings were taken in a spectrophotometer at 550 nm compared to a NaNO2 standard curve

(ranging 0.5 AM–25 AM) and the amount of nitric oxide release in the supernatant was measured

in each set (Sasaki et al., 1998).

DNA Fragementation

Cells (106 cells/200 Al) were resuspended in hypotonic lysis buffer [0.2% Triton X 100, 1 mM

Ethylene Diamine Tetraacetate (EDTA), pH 8.0] and centrifuged for 15 min at 1400 � g. The

supernatant containing small DNA fragments was collected and the cell pellet containing large pices

of DNA, cell debris was used for Diphenylamine (DPA) assay (Ochiai et al., 1997). Perchloric acid

(0.5 M) was added to the pellets containing uncut DNA (resuspended with 200 Al hypotonic lysis

buffer) and to the other half of the supernatant containing DNA fragments, and then 2 volumes of a

solution containing 0.088 M DPA, 98% (vol/vol) glacial acetic acid, 1.5% (vol/vol) sulfuric acid, 0.5%

(vol/vol) of 1.6% acetaldehyde solution were added. The samples were stored at 4 jC for 48 hrs. The

colorimetric reaction was quantified spectrophotometrically at 575 nm using a UV spectrophotometer.

The percentage of fragmentation was calculated as the ratio of DNA in the supernatant to the total

DNA.

Statistical analysis

Level of significance of all the parameters were determined by Student’s ‘t’ test.

Results

Hormone assay

Whether surgical removal of single adrenal gland leads to the reduction in serum cortisol level was

evident by cortisol hormone assay (Table 1).

Table 1

Changes in serum cortisol level after unilateral adrenalectomy

Days after adrenalectomy Cortisol level in serum (ng/ml)

Control Group Unilaterally Adrenalectomized Group

10 64.510 F 4.910 27.850 F 1.850

20 69.725 F 2.825 44.130 F 4.070

30 66.975 F 8.775 48.275 F 0.925

Results are expressed as mean F SD (p < 0.01).

Biochemical parameters

Effect of in vivo glucocorticoid depletion on random blood glucose level in rats

As glucocorticoid has immense effect on the carbohydrate metabolism particularly on blood

glucose level, liver glycogen synthesis and mobilization, estimation of random blood glucose level

may be useful to correlate the effect of in vivo GC depletion on carbohydrate metabolism. The

results showed significant increase (p < 0.025) in random blood glucose level compared to control

(Fig. 1).

Effect of in vivo glucocortcoid depletion on blood inorganic phosphate level

Estimation of inorganic phosphate level of blood was done since it may give an idea regarding

the status of phosphorylation-dephosphorylation mechanisms in the body. The results showed that

inorganic phosphate content of blood significantly increased (p < 0.0005) in adrenalectomized rats

(Fig. 2).

Effect of in vivo glucocorticoid depletion on serum protein level in rats

Adrenalectomy was found to be associated with dysproteinemia at least in the first phase although it

might be normalized later on suggesting that the GC deficiency also reduces inter-organ amino acid

mobilization. Results showed a significant decrease (p < 0.01) in serum protein level in adrenalecto-

mized rats with respect to control (Fig. 3).


Fig. 1. Effect of in vivo glucocorticoid depletion on random blood glucose level in rats. Results are expressed as mean F SD

(p < 0.025).

Fig. 3. Effect of in vivo glucocorticoid depletion on serum protein level in adrenalectomized rats. The results in this figure

represent the serum protein level (mean F SD) of control and adrenalectomized rats of three independent experiments

(p < 0.01).

Fig. 2. Effect of in vivo glucocorticoid depletion on blood inorganic phosphate level. Data are expressed as mean F SD.

Experiments were completed in triplicate (p < 0.0005).


Effect of in vivo glucocorticoid depletion on serum urea level in rats

As glucocorticoids intimately influence protein metabolism in the body, estimation of serum urea may

be an important factor to determine the extent of protein catabolism in the body during in vivo GC

depletion. Results showed a significant increase in serum urea level (p < 0.005) of adrenalectomized

rats compared to control (Fig. 4).

Effect of in vivo glucocorticoid depletion on glycogen content of liver in rats

Whether in vivo GC depletion may alter liver glycogen content was an important question. In order to

address this, the total glycogen content of liver in both control and ADX rats were determined. Results

showed that glycogen content of liver decreased significantly (p < 0.05) in adrenalectomized rats than

that of control (Fig. 5).

Hematological parameter

Blood samples were collected from the retroorbital plexus of previously adrenalectomized rats at

different time intervals and hematological parameters were determined (Table 2).

Alteration in Immune Response

Intracellular killing capacity of rat splenic macrophages in glucocorticoid insufficiency after

adrenalectomy

During phagocytosis specialized cells such as splenic macrophages take up and degrade bacteria that

need to be eliminated from the body. Whether adrenalectomy can lead to any change in the aforesaid


Fig. 4. Effect of in vivo glucocorticoid depletion on serum urea level in adrenalectomized rats. Results are expressed as meanFSD of triplicate experiments (p < 0.005).

Fig. 5. Effect of in vivo glucocorticoid depletion on glycogen content of liver in rats. Results in the figure represent the liver

glycogen content (mean F SD) of control and adrenalectomized rats of three independent experiments (p < 0.05).


function of macrophages needs to be clarified. The kinetic profile obtained showed that intracellular killing

capacity of splenic macrophages recovered from control and unilaterally adrenalectomized rats after 10th

and 20th days of adrenalectomy showed no significant alteration (p > 0.05). However function of splenic

Table 2

Effect of in vivo glucorticoid depletion on Hemoglobin (Hb) concentration, Haematocrit value (Hct), Total count (TC) and

Differential count (DC) of leukocytes

Haematological Days after adrenalectomy

parameters 10 20 30(mean F SD)

Control ADX Control ADX Control ADX

Hemoglobin

(gm/100 ml)

11.0 F 1.414 12.0 F 1.414 11.5 F 0.707 13.0 F 1.414 10.5 F 0.707 12.5 F 0.707

Hematocrit value

(in percentage)

35.0 F 1.414 37.0 F 1.414 34.0 F 2.828 39.0 F 1.414 35.5 F 0.707 42.0 F 1.414

Total cell count

(cells/Al)6100 F 141.4 6700 F 282.8 6350 F 353.6 7000 F 282.8 6350 F 212.1 7500 F 424.3

Differential count (in percentage)

N = Neutrophil N� 42.5 F 0.71 43.5 F 0.71 46.5 F 0.71 43.0 F 1.40 43.5 F 0.71 42.5 F 0.71

E = Eosinophil E� 2.5 F 0.71 3.5 F 0.71 3.5 F 0.71 4.0 F 0.0 1.5 F 0.71 2.5 F 0.71

B = Basophil B-� � � � � �L = Lymphocyte L� 29.0 F 1.41 34.5 F 0.71 29.5 F 0.71 37.0 F 1.41 28.5 F 0.71 36.5 F 0.71

M = Monocyte M� 21.5 F 0.71 16.0 F 0.71 22.5 F 2.12 17.0 F 1.41 23.5 F 2.12 19.0 F 1.41

Effect of adrenal corticosteroid insufficiency due to adrenalectomy on the hematological parameters in Swiss albino rat.

The results are expressed as mean F SD of triplicate experiments.


macrophages recovered from rats after 30 days of adrenalectomy showed altered response. It was observed

that viability of bacteria gradually decreases in control cells with time, whereas in case of ADX rat the

viability of S. aureus gradually increases which indicates the splenic macrophages in hormonal

insufficiency cannot ingest S.aureus properly so more bacteria were left viable by them (p < 0.01)

(Fig. 6).

Nitric oxide release from rat splenic macrophages in glucocorticoid insufficiency after adrenalectomy

A number of antimicrobial and cytotoxic substances released by activated macrophages are

responsible for intracellular destruction of phagocytosed microorganism, nitric oxide (NO) itself has

potent antimicrobial activity. Therefore a modulation in the release of NO could designate a decline in

the animal’s ability to defend itself from microbes. It was observed from the spectrophotometric readings

that the amount of NO released by the macrophages recovered after 10, 20, 30 days of adrenalectomy

was reduced with respect to control in both LPS nonstimulated and LPS stimulated cases (p < 0.05)

(Fig. 7).

Fig. 6. Intracellular killing capacity of splenic macrophages recorded from endogenously glucocorticoid depleted rat.

Intracellular killing was expressed as the percentage decrease in initial number of viable intracellular bacteria. The result in this

figure represents the decrease (%) in viability of S. aureus from different groups of three independent experiments (p < 0.01).

Fig. 7. Effect on in vivo glucocorticoid depletion on nitric oxide release from the rat splenic macrophages. The results in this

figure represent the nitric oxide released (in AM) and values were expressed as mean F SD of three independent experiments

(p < 0.05).


Effect of adrenalectomy on the functional integrity of rat splenic macrophages-as determined by

fragmentation of DNA

The effect of adrenalectomy on the functional integrity of splenic macrophages may demonstrate

particularly from its effect on DNA fragmentation to deduce whether adrenal hormone insufficiency may

lead to apoptosis. In our study the percentage of DNA fragmentation in splenic macrophages after

adrenalectomy showed significant (p < 0.005) reduction compared to control (Fig. 8).

Fig. 8. Effect of in vivo glucocorticoid depletion on the functional integrity of splenic macrophages as determined by

fragmentation of DNA. Results in this figure represent the extent of DNA fragmentation (in percentage) of rat splenic

macrophages recovered from control and adrenalectomized rats (p < 0.005).


Discussion

To determine the importance of adrenal corticosteroid on the effects of metabolic activity and immune

system we investigated changes in few biochemical and immunological parameters in intact and

unilaterally adrenalectomized rats. Adrenalectomy produced an increase in the blood glucose level.

Since adrenalectomy removes adrenal medulla as well as adrenal cortex and so eliminates the principal

source of epinephrine, hence in this case the effect of epinephrine on blood glucose regulation may also

be decreased. Adrenalectomy was also known to produce a decrease in plasma insulin (Kawai and

Kuzuya, 1977) and so the blood glucose lowering effect of insulin in this case cannot be avoided. As

adrenalectomy reduces the amount of glucocorticoid it may be inappropriate for a number of metabolic

reaction to occur indicating the impairment of permissive action of GC. So requirement of glucagons,

catecholamines to exert their calorigenic effects has not been permitted in the paucity of GC. Hence

plasma glucose lowering effect of insulin is greatly hampered in adrenal insufficiency and it can be

suggested that peripheral glucose utilization including that in the liver is also decreased. As a result there

is also significantly less glycogen deposition seen in liver of adrenalectomized rats. But the possible

mechanism remain unclear.

In absence of glucocorticoid hormone, glucocorticoid receptor remains anchored in the cytoplasm as a

large protein aggregate complexed with an inhibitor protein. In this situation, the receptor cannot interact

with target genes, hence no transcriptional activation occurs. After adrenalectomy decreased plasma

level of GC may be insufficient to release the glucocorticoid receptor from the cytoplasmic anchorage

and as a result it is not allowed to enter the target cell nucleus and hence the action of gloucocorticoid on

target cell is prevented.

Glycogen deposition in the liver is also significantly reduced in adrenalectomized rats which is

ultimately reflected in higher blood glucose level. Moreover, a depletion of GC level causes increased

hepatic glycogen deposition.

Blood inorganic phosphate level was also greatly enhanced in animals with adrenal insufficiency

indicating possible inhibition of phosphorylation. Since in vivo, many enzyme contributed metabolic

regulation depends on reversible phosphorylation and dephosphorylation it may be assumed that

inorganic phosphate was not properly utilized in enzyme phosphorylation, which led to higher blood

inorganic phosphate level after adrenalectomy. Although 30 days of unilateral adrenalectomy the

cortisol level returns to 65% of their controls levels from the rest of the hypertrophied adrenal gland, but

it was also reported that during passage of blood through liver about 50% each of glucagons,

norepinephrine, cortisol was degraded. So it may be inappropriate for a number of metabolic reactions

to occur indicating the impairment of permissive action of glucocorticoid. Moreover the functions of

liver cells are also controlled by circulating substances, products and hormones through blood and

intrahepatically formed mediators. The hepatocytes sense the hormone gradients via respective hormone

receptors which are distributed homogeneously in liver play a major role in the zonal expression of

different metabolizing enzymes. So at 30 days after adrenalectomy the increased phosphate/ urea level

or decreased serum protein level may be due to alteration of zonal distribution of enzymes (like

glycogen synthase, glycogen phosphorylase, glucose-6-phosphatase, phosphoenol pyruvate carboxy

kinase etc). As a result phosphate and amino acids are not properly utilized by these enzymes which

need to be phosphorylated or dephosphorylated for their action and vice versa or in other words

phosphate availability to these enzymes may decrease which ultimately resulted in enhanced serum

phosphate level.


Adrenalectomy produced a definite disturbance in the protein synthesizing function of liver

(Kudalaenko and Levshin, 1977). This result is in accordance with significant decrease in level of

serum protein in the adrenalectomized rats. It may also be due to lessened accumulation of free amino

acids in the blood of adrenalectomized animals (Stele, 1975) or due to the excessive protein catabolism

triggered by adrenalectomy resulting in depleted levels of GC in blood. This increased protein

catabolism in the metabolic scenario is also reflected in the significantly higher levels of serum urea

seen in the adrenalectomized rats during the study. Serum protein level was found decreased at 30 days

after adrenalectomy. It is unknown to us whether this effect is achieved by directly inhibiting release of

gluconeogenic amino acid from peripheral tissues such as skeletal muscles or either by altering hepatic

gluconeogenic enzymes (such as glucose-6-phosphatase or phosphoenol pyruvate carboxy kinase) or by

degrading serum proteins.

Since we obtained less protein in the serum of adrenalectomized rats compared to control, it may

possibly indicate the utilization of these amino acids in gluconeogenesis in liver or can be conversion to

urea as a result of which the blood glucose and urea concentration were high in adrenalectomized rats.

Since glucose and urea are the major non-electrolytes of plasma their contribution to plasma osmolality

may become quite large in high blood glucose and urea level; however, we have not tested this so far in

our study.

In adrenalectomized rats elevated hematocrit (Hct) value and increased Hemologbin (Hb) concen-

tration indicate erythrocyte release from spleen during corticosteroid insufficiency. Since Hb content of

blood indicates the status of normal red cell metabolism, it indirectly gives a gross idea about the

erythrocyte control and functioning of the erythropoietic machinery in the blood. Hct indicates the

stability of erythrocyte population in blood in terms of their total number. We therefore suggest that,

splenic contraction occurring in adrenalectomized rats may cause increased Hct and Hb concentration.

However, the increases in Hct and Hb were not accompanied by an increase in total plasma protein

content, which counters the cause being hemoconcentration due to fluid leaving the vascular system by

increased filtration. This indicates that the cause may be splenic contraction which is further supported

by elevated level of Hct and Hb in adrenalectomized groups.

Total leukocyte count was increased in adrenalectomized rats indicating that the generation of blood

cells is partly regulated by circulating glucocorticoid level. From the differential count numbers of

peripheral lymphocytes was increased after adrenal corticosteroid insufficiency which may be inter-

preted as immunopotentiation in absence of circulating GC due to adrenalectomy. These findings

support the hypothesis that the endogenous glucocorticoid may play a role in maintaining the metabolic

activities of rats.

Any modulation of the function of the immune system may cause inhibition in the capacity of the host

against nonself, greater susceptibility to infectious agents and pathogens. However, partial depletion of

endogenous GC by adrenalectomy also abolishes ingestion capacity of splenic macrophages as it can be

seen from the intracellular killing assay. Since cells recovered from adrenalectomized rats (after 30 days)

are not active enough or somehow less potent to kill S. aureus efficiently, we obtain more viable bacteria

in the supernatant. Normally GC inhibits the growth and activation of macrophages (in vivo) by reducing

Granulocyte Macrophage Colony Stimulating Factor (GMCSF) secretion and also block monocyte to

macrophage differentiation (Sakai et al., 1999). Since at 30 days after unilateral adrenalectomy the rest of

the adrenal grand ( hypertrophied) may replenish the GC, which may cause proportionate decrease in

macrophage function. As macrophages mature there is progressive rise in lysosomes and their hydrolytic

component. Adrenal hormone insufficiency may also lead to immaturation of macrophages in vivo in the


initial stage of GC depletion and it can be assumed that they have lesser lysosomal content and hence are

less capable of digesting bacteria. However, in our study no focus has been made to elucidate the enzyme

system interaction for microbial killing after adrenalectomy.

Nitric oxide released from splenic macrophages of adrenalectomized rat are less than that of control

macrophages even after stimulation with bacterial lipopolysaccharide (LPS). When macrophages are

activated with bacterial cell wall they begin to express high level of nitric oxide synthase which oxidizes

L-arginine to yield citrulline and nitric oxide (NO). NO itself has potent antimicrobial activity and it can

also combine with superoxide anion to yield even more potent antimicrobial substances (Tian and

Lawrence, 1996). It was reported that adrenalectomy not only reduces the secretion of GC from the

adrenal gland but also results in less secretion of macrophage migration inhibitory factor (MIF) from the

cells of the adrenal cortex (Thierry et al., 1995). MIF is a known costimulator of NO release (Nishihara

et al., 2000). Due to adrenalectomy MIF also depleted which causes reduced NO release even after

stimulation with LPS.

DNA fragmentation assay shows a lesser degree of fragmentation of splenic macrophages recovered

after adrenalectomy, which indicates apoptotic death of cells in this group decreases. Apoptosis includes

pronounced decrease in cell volume, membrane blebbing, condensation of the cytoplasm and chromatin

and fragmentation of DNA. Since splenic macrophage recovered 20 days after adrenalectomy have less

contact with endogenous GC, so glucocorticoid induced death may be prevented as well as differen-

tiation of monocytes into tissue fixed macrophages (in spleen) is also hampered due to less

glucocorticoid level in circulation after unilateral adrenalectomy.

Thus partially glucocorticoid depleted rats show reduced intracellular killing capacity, NO release,

improper cell maturation that heightens the probability of infection. However, the exact mechanism,

by which adrenalectomy causes the aforesaid immunomodulation is unknown and needs further

investigation.

Acknowledgement

The corresponding author (Dr. B. Bishayi) expresses his gratitude to the University Grants

Commission (U.G.C.), Govt. of India, New Delhi, India for providing financial assistance in this study.

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Metabolic and immunological responses associated with in vivo glucocorticoid depletion by...

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