Metabolic effects of cortisol in man—Studies with somatostatin

Metabolic

Effects

of Cortisol

D. G. Johnston,

in Man-Studies

A. Gill, H. @rskov, G. F. Batstone,

With Somatostatin

and K. G. M. M. Alberti

The metabolic effects of chronic hypercortisolaemie were studied by administration of tetracosactrin-depot, 1 mg I.M. daily for 36-60 hr to normal subjects. Partial insulin and glucagon deficiency were induced at the end of the period by infusion of somatostatin, 100 /.rg/h for 210 min. Tetracosactrin alone induced a three fold rise in basal serum cortisol levels and fasting blood glucose concentration rose from 5.2 + 0.2 to 7.2 + 0.2 mmole/l (p < 0.01) with a rise in fasting serum insulin from 5.2 f 1.2 to 13.1 +_ 1.9 mU/I (p < 0.02). Concentrations of the gluconeogenic precursors lactate, pyruvate and alanine were also raised, but non-esterified fatty acid, glycerol and ketone body levels were unchanged. Somatostatin infusion caused a 30%~50% decrease in serum insulin and a 20%-60% decrease in plasma glucagon concentrations both before and after tetracosactrin administration. A similar rise in blood glucose concentration, relative to the saline control, occurred over the period of somatostatin infusion both with and without elevated cortisol levels. However, prior tetracosactrin administration caused a 100% greater rise in blood ketone body concentrations during infusion of somatostatin than was seen in the euadrenal state, despite similar plasma NEFA concentrations. Hypercortisolaemia causes hyperglycaemia and elevated gluconeogenic precursor concentrations but the associated rise in serum insulin concentrations limits lipolysis and ketosis. In insulin deficiency, a ketotic effect of glucocorticoid excess is evident which may be independent of lipolysis and occurs despite concurrent glucagon deficiency. These catabolic actions of cortisol are likely to be of major importance in the metabolic

response to stress.

C

ORTISOL SECRETION is increased in conditions of stress together with secretion of the other catabolic hormones, glucagon, catecholamines and growth hormone.‘-’ In such circumstances, insulin secretion is diminished as a result of the inhibitory effect of catecholamines on insulin release.4 The metabolic response to stress which may persist for weeks in severely injured patients consists of increased glycogenolysis and gluconeogenesis, increased protein catabolism and a stimulation of lipid mobilisation with enhanced hepatic ketogenesis.5 Insulin deficiency is probably important in this metabolic adaptation but the role of individual counter-regulatory hormones is uncertain. This study was therefore designed to examine the metabolic sequelae of cortisol hypersecretion in subjects with normal insulin secretion and in subjects rendered partially insulin-deficient by infusion of somatostatin. Simultaneous suppression of glucagon secretion by somatostatin allowed separation of the direct effects of cortisol and those mediated through an increase in glucagon secretion.

From the Endocrine Unit and Department of Clinical Biochemistry and Metabolic Medicine, Royal Victoria Infirmary, Newcastle upon Tyne NEI 4LP, Birmingham General Hospital, Birmingham, U.K.. Institute of Experimental Clinical Research, University of Aarhus, Denmark, and the Salisbury Royal Infirmary. Salisbury, Wiltshire. U.K. Receivedfor publication November 24. 1980. Supported in part by a Grant from the British Diabetic Association. D. G. Johnston was the recipient of a Medical Research Council Fellowship. Address reprint requests to Dr. D. C. Johnston, Endocrine Unit, Royal Victoria Infirmary, Newcastle upon Tyne, U.K. 0 1982 by Grune & Stratton, Inc. 0026-0495/s2/3I04~01$01.00/0

312

PROTOCOL

Five normal subjects, aged 21-40 years, mean f SEM percent ideal body weight 106 + 3, were studied after an overnight (12 hr) fast. Dietary carbohydrate intake was in excess of 300 g daily for at least 48 hr before all studies and no subject was on any drugs. Intravenous teflon cannulae were positioned in both antecubital fossae between 0800 hr and 0830 hr and subjects remained recumbent for the duration of the test. Through one cannula subjects received continuous intravenous infusions on separate days of (1) 0.154 mole/l sodium chloride (30-50 ml) for 210 min, and (2) synthetic linear somatostatin (Ferring), 100 pg/hr, in 0.154 mole/l saline (30-50 ml) for 210 min. The contralateral arm was used for blood sampling. Two basal blood samples were taken 30 min after cannulation at which time continuous infusion was commenced. Further blood samples were taken at 30 min intervals for 4 hr. During somatostatin infusion, additional samples were taken 5 and 15 min after the start and finish of the infusion. Depot-tetracosactrin (Synacthen), 1 mg intramuscularly, was then administered daily at 2200 hr for 3 days and further saline and somatostatin infusion studies performed in random order on the mornings after the second and third tetracosactrin dose. Blood for cortisol estimation was taken by venepuncture at 1000 hr on the morning after the first tetracosactrin injection, The nature and purpose of the study was explained to all subjects and the study was approved by the Southampton General Hospital Ethical Committee. MATERIALS

AND

METHODS

Blood for glucose, 3-hydroxybutyrate, lactate, pyruvate, glycerol and alanine estimation was taken in ice-cold 5% (v/v) perchloric

Metabolism,

Vol. 31, No. 4 (April), 1982

313

CORTISOL AND METABOLISM

acid and assayed by automated fluorimetric enzymatic methods.6 Acetoacetate was measured by a manual spectrophotometric method on perchlorate samples.’ Serum triglyceride was measured as glycerol after hydrolysis’ and plasma non-esterified fatty acids (NEFA) by a radio-active cobalt method? Serum insulin’0 and growth hormone” were measured by double antibody radioimmunoassay. Blood samples (2.25 ml) for glucagon assay were collected into 0.25 ml aprotinin (2,500 K.I.U.) containing 25 pmole EDTA and plasma glucagon measured by wick chromatography. Serum cortisol was measured by competitive protein binding.‘r Statistical analysis was performed using Student’s t test for paired data.

RESULTS

Fasting Hormone and Metabolite Concentrations Depot-tetracosactrin administration caused a threefold rise in serum cortisol concentrations, evident within 12 hr (mean f SEM serum cortisol 12 hr after first injection 1202 + 90 nmole/l) which was sustained throughout the study period (Table 1). Chronic (36-60 hr) hypercortisolaemia produced a rise from basal in fasting blood glucose concentration (5.2 + 0.2 to 7.2 k 0.2 mmole/l, p < 0.01) and fasting serum insulin levels (5.2 + 1.2 to 13.1 + 1.9 mu/l, p < 0.02 compared with values obtained before tetracosactrin administration). Concentrations of the gluconeogenic precursors lactate, pyruvate and alanine were also raised (Table l), but the 1actate:pyruvate ratio was unchanged. Basal blood ketone body, glycerol and plasma NEFA concentrations were unchanged (Table 1) but serum triglyceride levels decreased from 1.24 f 0.40 to 0.68 t 0.19 mmole/l (p < 0.05). Plasma glucagon and serum growth hormone concentrations were unaltered (Table 1).

secretion observed during saline infusions were suppressed by somatostatin before and after tetracosactrin (Table 1). A rebound in insulin, glucagon and growth hormone secretion was seen on cessation of somatostatin infusion. Somatostatin produced a rise in blood glucose concentration, significant from 120 min compared to control saline infusion, before tetracosactrin administration (Fig. 1). At 210 min the mean change in blood glucose was +0.6 + 0.3 mmole/l for somatostatin, -0.3 * 0.1 mmole/l for saline, p < 0.05. After tetracosactrin, blood glucose concentrations were also elevated by somatostatin, significantly compared to saline infusion from 90 min onwards, although this difference was less marked. Concentrations of the gluconeogenic precursors, lactate, pyruvate and alanine were elevated after tetracosactrin and remained so throughout the saline infusion period. Somatostatin had no effect on gluconeogenic precursor concentrations before or after tetracosactrin (Table 1). Tetracosactrin caused a rise in plasma NEFA levels during saline studies (Fig. 2). Somatostatin infusion alone caused a minor rise in plasma NEFA concentrations in the euadrenal state. After tetracosactrin, somatostatin caused no further increase. Blood concentrations of ketone bodies did not alter significantly during the saline infusions. Somatostatin alone produced a minor rise in ketone body concentrations significant compared to saline only at 210 min (0.23 _t 0.1 1 vs 0.14 t 0.06 mmolejl, p < 0.05). After tetracosactrin ketone body concentrations were markedly elevated from 60 min onwards during somatostatin infusion.

Infusion Studies Serum cortisol levels in untreated subjects fell over the period of saline infusion, reflecting the normal diurnal variation of cortisol (Table 1). Infusion of somatostatin did not alter this pattern. After tetracosactrin, cortisol levels were similarly elevated throughout the study period in somatostatin and saline infused subjects. Somatostatin infusion produced a 30%-50% decrease in circulating serum insulin concentrations, both before and after tetracosactrin administration (Fig. 1). Insulin levels during infusion of both saline and somatostatin were higher after tetracosactrin than before. Plasma glucagon concentrations after tetracosactrin administration were also elevated at certain times in the saline study (Table 1). Somatostatin produced a 20%-60% decrease in plasma glucagon levels. The spontaneous surges in growth hormone

DISCUSSION

Cortisol exerts its metabolic activity by regulating DNA, RNA and subsequent enzyme protein synthesis14 unlike glucagon and the catecholamines which have a rapid action on carbohydrate and lipid metabolism. This mechanism of action requires several hours before metabolic effects are seen in vitro. The importance of cortisol in stress should however not be underestimated as secretion in injured patients may remain elevated for days or weeks after the initial injury.’ We therefore chose to examine the metabolic consequences of 36-60 hr cortisol hypersecretion. Hypercortisolaemia was induced by administration of a synthetic ACTH-like peptide” and serum coristol concentrations were comparable to those obtained in injured patients. Although ACTH itself may have direct effects of intermediary metabolism, particularly on lipolysis,‘6 these in vitro actions are seen at pharma-

0.07 0.06 * 0.0 1 0.24 + 0.03 0.06 + 0.01 1.08 t 0.37 252 & 53 40 +

+ 0.19 0.10’ f 0.02 0.37* + 0.02 0.05 * 0.01 O.JZ* k 0.28 1.237* * 74 50 k 18

0.11 o.ost + 0.01 0.38t + 0.04 0.06 k 0.01 0.68t + 0.19 1,179t t 97 49 f 14 0.55 + 0.07

+

0.03

0.07

f

0.01

0.26

+

0.03

0.04 +

0.01

1.19

f

0.30

428

+

35

52

f

18

0.43

zk

0.09

0.09

0.07

+

0.01

0.25

k

0.04

0.06

k

0.01

1.24

k

0.40

441

+

40

38

*

15

0.88 +

0.44

Blood Pyruvate

mmole/l

Blood Alanine

mmole/l

Blood Glycerol

mmole/l

Serum Cortisol

mmoletl

*

*

* 0.01

k 0.01 0.26 0.02 0.07 f 0.01 0.97 + 0.26 190 f 33 29 k 8

0.01 0.23 t 0.04 0.07 f 0.01 1.14 * 0.4 1 231 k 37 43 zt 12

+ 0.01 0.31 + 0.02 0.08 + 0.01 0.88 + 0.39 1,329. * 66 37 + 14

* 0.01 0.33t t 0.04 0.07 + 0.01 0.79 t 0.23 1,259t + 79

6’3 t 16

* 0.01 0.26 f 0.03 0.06 + 0.01 1.03 + 0.30 160 -t 20 26 8

= values during somatostatin infusion significantly different before vs after tetracosactrin administration, p < 0.05.

SRIF = somatostatin infusion.

0.28

0.02 0.18 0.09

0.06

0.06

0.11

0.31

h administration of tetracosactrin-depot.

* +

+ *

t

k

*

1.05

0.30

0.61 0.44

0.37

0.40

0.49

17

5’3 +

90

+

1,ZOJt

0.23

*

0.75

0.07

0.03

k

0.08t

0.10

0.79 &

*

0.31

0.31

0.05

0.05 *

0.07”

0.08

0.05

k

0.01

0.05

0.06

0.06

0.09

0.01

*

0.73t

f

+

+

+

SRIF

0.04

+

0.38

12

+

31

103

*

1.372’

0.45

f

0.93

0.01

*

0.08

0.02

0.01

f

O.OJ*

0.05

+

0.73*

0.59

Saline

SRIF 0.64

Saline

SRIF 0.72

Saline 0.75t

After Tetracosactrin

210 min

SRIF

Before Tetracosactrin

and Saline Before and After Tetracosactrin

0.59 +

After Tetracosactrin

120 min

t = values during saline infusion significantly different before vs after tetracosactrin administration, p < 0.05.

l

Statistical analysis by student’s t test for paired data.

Values are shown as mean + SEM before and after 36-60

“g/t

Serum Growth Hormone

“g/l

Plasma Glucagon

mmole/l

Berum Triglyceride

18

0.64 +

1.06’

0.82t *

0.70

0.68 *

mmolejl

Blood Lactate

Saline

SRIF

Before Tetracosactrin

Saline

After Tetracosactrin

During Infusion of Somatostatin

SRIF

0 min

Concentrations

Saline

Metabolitea Hormone

Before Tetracosactrin

Table 1. Hormone and Metabolite

0.04

+

0.42

4

t

41

30

*

238

0.39

f

1.10

0.01

f

0.06

0.04

+

0.25

0.01

k

0.05

0.04

+

0.58

Saline

1.98

2.36 +

17

*

43

28

+

227

0.27

+

0.95

0.01

+

0.05

0.04

*

0.27

0.01

+

0.06

0.05

+

0.65

SRIF

Before Tetracosactrin

Administration

0.37

*

1.15

11

k

69

88

k

1,099t

0.22

*

0.74

0.01

k

0.06

0.04

k

0.3 1

0.01

*

O.OJt

0.10

k

0.74

Saline

1.27

+

1.91

13

+

59

64

_c

1.328.

0.44

k

0.89

0.01

f

0.06

0.02

*

0.32

0.01

t

0.08’

0.06

*

0.78.

SRIF

After Tetracosactrin

240 min

315

CORTISOL AND METABOLISM

Swum (m”,I)

2o -_

I

20-

insulin

10-

10 -

Fig. 1. Serum insulin and blood glucose response to saline and somatostatin infusions. 6 saline infusion, n saline infusion after 36-60 hr tetracosactrin-depot, 0 somatostatin infusion, 0 somatostatin infusion after 36-66 hr tetracosactrin-depot. Statistical analysis was performed using Student’s t test for paired data. t saline infusions significant at 5% level or less, before versus after tetracosactrin, l somatostatin infusions significant at 5% level or less, before vs after tatracosactrin. All values shown as the mean + SEM.

O-

0'

a

8

Blood glucose (mmolll)

~***“”

u*

0

0

I

120 Min

cological concentrations and there is no evidence for a major direct metabolic effect of ACTH in intact man. A similar moderate elevation in fasting blood glucose concentration to that observed in this study, has been reported after short-term administration of glucocorticoid preparations to normal and diseased subGlucocorticoids decrease peripheral uptake

l

l

l

I

I

120

*

x

***

I

,

210 240

Min

of glucose by several tissues,‘&” and this may be the major mechanism for production of hyperglycaemia in normal man.23 In addition there is increased activity of several key gluconeogenic enzymes in the liver resulting in an increased capacity for gluconeogenesis24 and increased gluconeogenic substrate supply from the periphery in the form of lactate, pyruvate and ala-

jects,17,18,19

Blood total ketone bodies

I

(mmol/lJ 0.25 -

OL 1.50

r

Plasma NEFA (mmolllj

o.75_&

*L

OL L

0

I

120 Min

1

I

210 240

I

0

1

120 Min

I

210

,

240

Fig. 2. Blood ketone and plesma NEFA response to saline and somatostetin infusions. “Total ketone bodies” refers to the sum of 3-hydroxybutyrate and acetoacetate concentrations. Symbols and conditions as for Fig. 1.

316

JOHNSTON ET AL.

nine.25 Studies in diabetic man suggest that both increased glucose production and decreased glucose utilisation are operative in production of the hyperglycaemic action of cortisol.** The hyperinsulinaemia observed probably derives from the elevation in blood glucose concentrations, although a direct effect of cortisol on pancreatic insulin secretion has been demonstrated in vitro.26 The combination of hyperglycaemia with hyperinsulinaemia suggests that insulin resistance is also a major result of chronic cortisol excess. Plasma NEFA concentrations were elevated only slightly by tetracosactrin. A direct lipolytic action of glucocorticoids has been demonstrated in vitro in addition to an action potentiating the lipolytic effect of other hormones.*’ In vivo however this action is limited by compensatory hyperinsulinaemia resulting in the relatively small changes in plasma NEFA seen in our subjects. Lipolysis is exquisitively sensitive to small changes in circulating insulin concentrations*’ and in Cushing’s syndrome which is also associated with hyperinsulinaemia, plasma NEFA concentrations are normal and NEFA turnover, expressed in terms of body weight, may indeed be decreased.29 Despite the small increase in plasma NEFA seen in our subjects, basal blood ketone body concentrations and levels during the infusion of saline were unaltered by cortisol excess. This restriction on ketogenesis may also result from the elevation in serum insulin concentrations. In vitro evidence suggests that basal ketogenesis is determined by the fasting insulin: glucagon ratio.30 A glucocorticoid-induced rise in plasma glucagon levels has been previously described,” but in our study the relative rise in glucagon was less than that for insulin. Thus cortisol-induced hyperinsulinaemia relative to glucagon may restrict ketosis in hypercortisolaemic states. The fall in serum triglyceride concentration with hypercortisolaemia has been noted previously in rabbits in which the phase persists for 48 hr and is followed in 5 days by hyperlipidaemia with the appearance in plasma of abnormally large very low density lipoprotein (VLDL) molecules.3’ The biphasic response has also been noticed in diseased man given pharmacological doses of exogenous glucorticoid.32,33 The mechanism of the initial decrease in serum triglyceride concentration is uncertain but suppression of hepatic lipoprotein secretion has been suggested34

although there is also circumstantial evidence for an effect on peripheral triglyceride uptake. The dose of somatostatin infused in this study produced a partial insulin deficiency with basal levels suppressed by 30%5076, comparable to the degree of insulin deficiency seen in the early phase of injury. Glucagon secretion was comparably suppressed. An initial fall in blood glucose concentration with high doses of somatostatin has been consistently reported and reflects glucagon deficiency.35-38 In agreement with other studies, prolonged somatostatin infusion resulted in hyperglycaemia, previously shown to be secondary to both increased production and decreased utilisation of glucose.39,40 A small hyperglycaemic response during somatostatin administration was also observed in the hypercortisolaemic state in our study despite fourfold higher serum insulin values, emphasising the direct importance of cortisol in the chronic hyperglycaemic effect observed. Prolonged somatostatin-induced insulin-deficiency resulted in a minor rise in plasma NEFA and blood ketone body concentrations and Wahren and coworkers have demonstrated a somatostatin-induced increase in splanchnic NEFA uptake and ketone body output.38 Despite similar plasma NEFA levels during somatostatin infusion before and after tetracosactrin, a 100% greater rise in blood ketone body concentrations was observed in the hypercortisolaemic state. A ketogenie action of glucocorticoids in insulin-deficiency was first demonstrated in elegant experiments with hypophysectomised and adrenalectomised diabetic animals given replacement ACTH or glucocorticoid.4’,42 Subsequent experiments in diabetic man have also suggested a ketogenic action of cortiso1.43.44 Our study confirms this ketogenic effect in the presence of only partial insulin deficiency and suggests that the action is independent of lipolysis, as determined by plasma NEFA concentrations. Thus chronic elevation in plasma cortisol levels results in increased circulating levels of insulin, glucose and the gluconeogenic precursors. Infusion of somatostatin unmasks a ketotic effect of cortisol.

ACKNOWLEDGMENT We are grateful to Miss L. Hinks and Miss P. Smythe for technical assistance and to Ferring Pharmaceuticals who kindly supplied the somatostatin.

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factors

of diabetic

ketosis. Diabetes

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