A M E C H A N I S M OF T H E G L Y C O G E N O L Y T I C A C T I O N OF B A C T E R I A L E N D O T O X I N *
BY JAY P. SANFORD, M.D., JACK A. BARNETT,$ M.D., Am~
CORA GOTT
(From the Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas)
(Received for publication, February, 26, 1960)
Determination of the site of action of bacterial endotoxin has been the object of extensive investigation (1-6).
Hyperglycemia and depletion of liver glycogen are the most constant biochemical changes which have been observed to follow the injection of bacterial endotoxin (1-3). These changes have been assumed to represent a direct hepatotoxic action of endotoxin, implying that bacterial endotoxin is capable of poisoning a specific en- zyme system or systems that mediate hepatic glycogenesis. If these assumptions are correct, they constitute an extremely important due regarding the mechanism of action of endotoxin on an enzymatic level.
However, hyperglycemia and depletion of hepatic glycogen are also well defined consequences of epinephrine release. I t is conceivable that the disturbances in car- bohydrate metabolism in the liver may not be the result of a direct toxic action on hepatic enzyme systems, but rather the passive consequence of epinephrine release.
The hypothesis that an adrenergic mechanism may be involved in the action of endo- toxin is supported by a considerable body of indirect evidence, eked by Thomas (7).
On the basis of such evidence, he has suggested "it is possible that some, if not all of the effects are closely related to the adrenergie action..."; however, "the mechanism by which endotoxins produced the observed metabolic abnormalities requires much further study" (7).
The following studies were designed to test the hypothesis that the changes in hepatic glycogen content following the administration of bacterial endotoxins are mediated through epinephrine release rather than by a direct toxic action.
I t is concluded that sublethal quantities of bacterial endotoxin influence hepatic glycogen stores as a consequence of epinephrine release.
Methods and Materials
AnimaJs.--Male rats of the Sprague-Dawley strain, weighing 105 to 115 gm., obtained from Holtzman Farms, Houston, were maintained on a standard antibiotic-free laboratory diet and
* The work was carried out under a contract with the Office of the Surgeon General, De- partment of the Army (DA-49-007-MD-884).
:~ Work performed during tenure of a Post-Doctorate Fellowship (EF 8852) of the Na- tional Institutes of Health.
97
9 8 GLYCOGENOLYTIC ACTION OF BACTERIAL ENDOTOXIN
acclimatized at least 1 week at 25°C. before any experimental procedure. The mice used for determination of endotoxin toxicity were females of the Swiss-Webster strain, weighing 18 to 20 gin., obtained from Rockland Farms New City, New York. They were maintained under the same laboratory conditions.
Adrenalectomy.--Bilateral adrenalectomy was performed through a dorsal incision under anesthesia consisting of pentobarbital and ether. Following adrenalectomy, animals were maintained on one of the following regimens: Desoxycorticosterone acetate, 0.5 rag.subcu- taneously daily (Sehering Corporation, Bloomfield, New Jersey lot No.6T-A4-P58-769, DOCA ® in sesame oil, 5 rag. per ml.), or cortisone acetate, 0.1 mg. subcutaneously daily (Upjohn Company, Kalamazoo, Michigan, lot No. K M 010 FE). The time interval between adrenalectomy and study was 5 to 7 days. During this interval the adrenalectomized animals were placed on 0.9 per cent saline solution as drinking water.
Dietary Regimens.--Animals were maintained on a standard antibiotic-free laboratory diet for the first 3 days postadrenalectomy. Both the adrenalectomized and the normal control animals which were to be studied in the well nourished state then were fed by gastric tube
TABLE I
Characteristics of Bacterial Endotoxin
Toxicity Bacterial species Source Method of preparation (Mouse
LD~0, rag.) Aerobacter aerogenes
Shigdla flexneri 2A Salmonella typhimur~m Escherichia coli 0-111
Sanford Noyes
Difco:~ No. B25624 Sanford
Boivin*
Boivin*
Boivin
Acetone-killed whole organisms
1.84 0.24 0.50 5.0
* Prepared according to technique published in reference 8.
Difco Laboratories, Detroit.
twice daily for an additional 3 days to assure comparable food intake in both groups. The animals were either tube-fed a balanced high glucose diet or allowed the standard laboratory diet ad lib/t~m, then tube-fed a supplemental 3 ml. of saturated glucose solution. Both dietary regimens resulted in comparable concentrations of hepatic glycogen.
Animals that were to be studied under conditions of fasting had food withdrawn 96 hours prior to study. During this interval the fasted animals were allowed water ad libitum.
gndotoxin.--Endotoxins from a variety of Gram-negative bacteria were utilized. The characteristics of these endotoxin preparations are tabulated (Table I). The Boivin-type endotoxins were prepared according to the technique of Noyes et al. (8). The quantities of endotoxin administered were sublethal for normal rats and adrenalectomized rats maintained on the large doses of DOCA ®. I n most studies, the rats received an estimated 2.5 mouse LDs0 doses of endotoxin per 100 gin. of body weight.
Glycogen Assay.---On the morning of study, animals were tube-fed. Two hours later adren- alectomized and control animals received either endotoxin or saline by intraperitoneal injec- tion. The animals were sacrificed 60 minutes later by decapitation, the livers were quickly removed and glycogen determinations carried out according to the method of Good, Kramer, and Somogyi (9). Glycogen values are reported as milligrams of glycogen per gram of wet liver.
j . P. SANFORD, J. A. BARNETT~ AND C. GOTT 99 Statistical A nalysis.--Most experiments were replicated on several occasions with different lots of rats. The replicate experiments were combined and the mean values and standard errors of the means calculated for each procedure. Differences between the means were compared by the t test.
E X P E R I M E N T A L R E S U L T S
T h e effect of b a c t e r i a l e n d o t o x i n on the h e p a t i c glycogen c o n t e n t of we~l nourished n o r m a l a n d a d r e n a l e c t o m i z e d r a t s is p r e s e n t e d in T a b l e I I . T h e a d - m i n i s t r a t i o n of s u b l e t h a l q u a n t i t i e s of B o i v i n - t y p e e n d o t o x i n u n i f o r m l y de-
TABLE I I
Effect of Various Bacterial Endotoxins on the Hepatic Glycogen Content of Normal and Adrenalectomized, Fed Rats*
Sxperl- mental group
1
2 4 5
Bacterial species
A erobacter aerogenes Shigella flex-
neri 2A Salmonella
typhimurium E. coli O-111
Normal
Saline
n$ Mean =h S.X.M. n
mg./gm.
17 21.4 -4- 1.3 16 7 20.6 4- 0.9 7 8 28.6 4- 3.4 7 4 25.6 -4- 3.4 4
Hepatic glycogen content
Endotoxin
Mean -I- S.Z.M n
mg./t,n.
6.5 4- 1.1 23 6.9 4- 1.C 6 9.2 4- 1.C 8 12.4 4- 1.7 4
Adrenalectomized S ~ e
Mean -4- S.E.M.
rAg./g,n.
21.3 4- 2.1 21.2 4- 2.9 26.2 4- 1.8 22.7 4- 2.4
Endotoxin n Mean q- s.v..M
reg./gin.
26 17.9 4- 1A 6 27.9 4- 2.(
7 26.2 q- 3.(
5 23.4 4- 1.(
* Adrenalectomized rats were maintained on either desoxycorticosterone acetate or corti- sone. The quantity of endotoxin administered was a sublethal quantity, approximating 2.5 mouse LD~0 per 100 gm. of body weight. Hepatic glycogen was determined 60 minutes after endotoxin administration.
n, abbreviation for number of animals in a given experimental group.
creased t h e h e p a t i c glycogen c o n t e n t of n o r m a l well n o u r i s h e d rats. T o a s c e r t a i n w h e t h e r the response was confined to e n d o t o x i n d e r i v e d from a single b a c t e r i a l species, or w h e t h e r i n s t e a d endotoxins from different species could elicit t h e s a m e response, B o i v i n - t y p e endotoxins were p r e p a r e d from these b a c t e n a l species. I n all instances a similar r e d u c t i o n in h e p a t i c glycogen (30 to 38 p e r c e n t of control values) was observed. T h a t the m e t h o d of p r e p a r a t i o n of endo- toxin was n o t responsible for this effect could be inferred from the o b s e r v a t i o n t h a t killed whole organisms (Esckerich/a coli O-111) caused a c o m p a r a b l e de- pletion of liver glycogen (48 p e r c e n t of control values).
A second g r o u p of e x p e r i m e n t s was p e r f o r m e d on a d r e n a l e c t o m i z e d r a t s ( m a i n t a i n e d on e i t h e r cortisone or desoxycorticosterone acetate) in o r d e r to
100 G L Y C O G E N O L Y T I C A C T I O N OF B A C T E R I A L E N D O T O X I N
obviate the effects of release of epinephrine from the adrenal medulla. The liver glycogen content of the cortisone-maintained adrenalectomized rats was the same as those treated with D O C A ® ; and both of these groups had values for liver glycogen content quite comparable to those values observed in normal rats (normal, 21.4 rag. per gin. wet weight of liver; cortisone-maintained ad- renalectomized, 20.6 rag. per gm.; DOCA®-maintained adrenalectomized, 22.0 rag. per gin.). I n contrast to the decreases in hepatic glycogen observed in normal rats, no significant decrease in the mean hepatic glycogen content oc- curred following the administration of either Boivin-type endotoxin or killed whole organisms to adrenalectomized rats. Thus, adrenalectomy followed b y
Ex- perl-
men- Bacterial species tsl
group
1 A erobacter aerogenes 2 A erobactev
aerogenes
TABLE I I I
fleet of Bacterial Endotoxin on the Hepati~ Glycogen Content of Normal Fasted Rats*
Combined
Mean body weight Hepatic glycogen content
Saline Endotoxin
Original
gin.
103 110
e r ~ D~-
i pe, r ce~
25 31
10 7
17
M e a n -4- S.Z.M.
1.04 4- 0.26 0.95 4- 0.32
1.0 4- 0.2
n M e a n 4- S.E.M.
,ng./gm.
10 2.02 4- 0.39 8 3.58 -4- 0.70
18 3.04 -4- 0.41
rag./gm.
=0.05
<0.01
<0.001
* Food was withdrawn 96 hours prior to experiment. Animals were allowed water ad Hb~um. Approximately 2.5 mouse LD~ per 100 gin. body weight of a Boivin-type endotoxin was administered and hepatic glycogen content determined after 50 minutes.
adequate corticosteroid replacement abolished the hepatic glycogenolytic action of sublethal quantities of representative bacterial endotoxins.
The effect of bacterial endotoxin on the hepatic glycogen content of normal rats fasted for 96 hours is presented in Table I I I . This period of fasting resulted in a 25 to 30 per cent decrease in body weight. Fasting effected a reduction of hepatic glycogen content from a mean of 21.4 mg. per gin. of wet liver to 1.0 mg. per gm. of wet liver. I n contrast to the decreases in hepatic glycogen content effected b y the administration of bacterial endotoxin in well nourished rats, the injection of the same quantity and lot of endotoxin into fasted rats resulted in a highly significant increase in mean hepatic glycogen content from 1.0 mg.
per gin. of wet liver to 3.0 mg. per gm. of wet liver (P < 0.001). Thus, liver glycogen content m a y either rise or fall after endotoxin administration de- pending upon the antecedent diet.
These data exclude an hepatotoxic action of bacterial endotoxin either
J. P. SAN'FORD, J. A. BARNETT~ AND C. GOTT 101 through direct interference with glycogen synthesis or through direct accelera- tion of glycogen degradation. Our experiments differ in two fundamental aspects from those interpreted by Kun as demonstrating a direct action of endotoxin on glycogen synthesis. First, he did not examine the effects of antecedent diet. His animals received glucose 2 and 3 hours before the admin- istration of endotoxin. Our observations of well nourished animals are in accord with those of Kun and other investigators. The increase in hepatic glycogen content demonstrated in fasted rats following the administration of endotoxin necessitates an alternative explanation. Second, he administered extremely
T A B L E I V
Effect of Lethal Doses of Bacterial Endotoxins on Hepatic Glycogen of Normal and Adrenalectomized, Fed Rats*
~.xperi- mental group
1 2
3
4
Bacterial species
E. coli O-26 Shigella flez- neri 2A Shigdla .~x-
n ~ , / 2 A
E. coli 0-111
Hepatic glycogen content
Normal
Saline
n ~[ean -4- S.E.K.
reg.~gin.
7 34.6 4- 3.9 4 25.6 -4- 3.4 1 42.6 2 : 3 2 . 5
Endotoxin
n Mean 4- S.E.M, n
reg./gin.
4 1 3 . 4 4 - 3.2 7 6 4 . 2 4 - 0 . 4 4
2 24.5 1
2 11.6 2
A&enalectomized
Saline
Mean -4- S.E.M.
reg.~gin.
34.3 =t: 2.5
22.7 4- 2 . 4
4 3 . 7
3 8 . 9
Endotoxin
n Mean 4- s . x . ~ .
mg./~m.
6 1 4 . 1 4 - 1 . 9 6 4 . 2 -4- 0 . 4
2 16.0
2 16.1
* T h e q u a n t i t y of endotoxin administered a p p r o x i m a t e d 20 m o u s e LDt~ per 100 gm. b o d y weight.
large quantities of endotoxin. To reconcile this difference in experimental design, the effect of lethal doses of bacterial endotoxin was studied in rats receiving 20 mouse LDs0 doses of endotoxin per 100 gin. of body weight rather than 2.5 mouse LD60 doses per 100 gin. as in the earlier studies (Table IV).
Under these circumstances, adrenalectomy failed to abolish the fall in hapatic glycogen content.
DISCUSSION
These experiments confirm the observations that depletion in liver glycogen content is a constant consequence of endotoxin administration to normal well nourished rats. H o w e v e r , reduction in liver glycogen content is not an invari- able consequence of endotoxin administration. Indeed, the administration of bacterial endotoxin to normal rats subjected to fasting results in a highly
102 G L Y C O G E N O L Y T I C A C T I O N O F B A C T E R I A L E N D O T O X I N
significant increase in liver glycogen content. In view of the fact that liver glycogen may either rise or fall after endotoxin administration, depending upon the antecedent diet, to assume poisoning of a specific enzyme system or systems that mediate glycogen synthesis is not reasonable. An alternative mechanism is required.
A similar divergence in changes in liver glycogen was demonstrated by the Coris to follow the administration of epinephrine to well nourished rats as compared to rats subjected to fasting (10-12). Epinephrine produces hyper- glycemia and depletion of liver glycogen when administered to well nourished animals. In contrast, the sequence of events that follow epinephrine admin- istration to a fasted rat is: (a) a fall in muscle glycogen, (b) a rise in blood lactate, and (c) a rise in liver glycogen. Though temporally different, the simi- larity in changes in liver glycogen content observed following the administra- tion of either epinephrine or endotoxin under different nutritional circumstances suggested that the action of endotoxin on liver glycogen was mediated through the release of epinephrine.
The importance of this mechanism was confirmed by the demonstration that total adrenalectomy followed by corticosteroid replacement abolished the depletion in liver glycogen following the administration of sublethal quantities of bacterial endotoxin to well nourished rats.
While adrenalectomy did not abolish the glycogenolytic effect associated with the administration of extremely large doses of endotoxin such as used by Kun (1, 2), this glycogenolytic effect may have been a secondary manifesta- tion of prolonged anoxia secondary to the hypotension which accompanies the administration of such large doses of endotoxin, the result of stimulation of extra-adrenal medullary sources of catechol amines or of a direct hepatotoxic effect.
The data presented indicate that the derangement of carbohydrate metab- olism following sublethal quantities of endotoxin cannot represent a direct interference with glycogen synthesis or direct acceleration of glycogen degrada- tion since the content of liver glycogen may either rise or fall after endotoxin administration, depending upon the antecedent diet. The fact that total adrenalectomy followed by corticosteroid replacement abolished the depletion in liver glycogen following the administration of sublethal quantities of bacterial endotoxin to well nourished rats is interpreted as indicating that the derange- ment of carbohydrate metabolism following sublethal quantities of endotoxin represents the passive consequence of epinephrine release.
There is a considerable body of evidence, much of which is cited by Thomas, which suggests that bacterial endotoxin exerts a sympathomimetic effect through the release of epinephrine (7, 13-17). Reilly and coworkers first pro- posed that the sympathetic nervous system was primarily involved in the action of endotoxin (14). Indeed, the physiological and pathological resemblances
j . p, SANFORD, J. A. BARNETT, .ANTI) C. GOTT 103 between endotoxin and epinephrine shock are striking. The histological evidence that depletion of adrenal chromaffm substance occurs after endotoxin was directly confirmed by Egdahl, who demonstrated a significant release of catechol amines into adrenal venous blood following administration of sublethal quanti- ties of bacterial endotoxin of the Boivin type to dogs (16, 17). Pretreatment of animals with adrenergic and ganglionic blocking agents affords protection against endotoxic shock (18-20). On the other hand, the injection of epine- phrine or norepinephrine into the skin of rabbits Within 4 hours after an intra- venous injection of endotoxin resulted in extensive dermal hemorrhagic necrosis (21). The changes seen within the gastrointestinal tract of animals following lethal doses of epinephrine and norepinephrine are similar to those seen in the intestinal mucosa following shock secondary to bacterial endotoxin (22, 23).
An additional similarity in the actions of endotoxin and epinephrine is the interference with the migration of leucocytes noted by DeLaunay and asso- ciates and by Miles and Niven (24, 25).
Total sympathectomy does not protect animals from the lethal effects of endotoxin (20). Also, the development of tolerance to levarterenol is not asso- ciated with the acquisition of cross-resistance to bacterial endotoxin (26).
These observations suggest that while increased amounts of either epinephrine or norepinephrine may be deleterious in the course of endotoxic shock, neither epinephrine nor norepinephrine are of themselves the common denominator in the lethal action of bacterial endotoxin.
CONCLUSIONS
These experiments have demonstrated that liver glycogen may rise or fall after endotoxin administration, depending upon the antecedent diet and that total adrenalectomy followed by cordcosteroid replacement abolishes the glycogenolytic effect of sublethal doses of endotoxin.
It is concluded that the derangements of carbohydrate metabolism observed following the administration of sublethal quantities of bacterial endotoxin represent, not a direct hepatotoxic effect of endotoxin, but rather the passive consequence of epinephrine release.
The authors wish to express their appreciation to Dr. Donald W. Seldin and Dr. Marvin D. Siperstein for their helpful criticism.
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104 G L Y C O G E N O L Y T I C A C T I O N O F B A C T E R I A L E N D O T O X I N
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26. Unpublished observations.
