MECHANISMS OF ENDOTOXIN TOLERANCE IN RELATION TO CARBOHYDRATE METABOLISM

Transcription

1 MECHANISMS OF ENDOTOXIN TOLERANCE IN RELATION TO CARBOHYDRATE METABOLISM Tokuro FUKUDA AND Setsuko AKIYAMA* Department of Physiology, Chiba University School of Medicine, Chiba It is well documented that repeated injections of endotoxins elicit resistance not only to the fever-producing effect of these materials but also to their various toxic effects, such as lethal, adrenohemorrhagic, tumor-necrotizing effect and to the Shwartzman phenomena1) The resistance is also known to be non-specific to the kinds of endotoxin produced by various Gram-negative bacteria and the specific immunity in the classical sense seems to play no part1). Most of the works on this reduced susceptibility has been made on the fever response in rabbits and the term "tolerance" has been used for its description. However, the fever response to endotoxin is not a reliable index to its action especially in rabbits from the following two reasons. Firstly the tolerance is never complete and animals continue to react to injection of endotoxin with small but definite "minimal response" 1). Secondly, with toxic dose of endotoxin a hypothermia rather than a hyperthermia follows. Enough fever response to large doses of endotoxin rather indicates a lessening of intoxication2). In this respect the depletion of liver glycogen and the accompanying hypoglycemia in endotoxin intoxication which have been shown to be the critical factor for the induction of late prostration and fatal outcome (FUKUDA2)) seem to be the most reliable index to the endotoxin intoxication. With this criterion it was shown here that the tolerance against endotoxin intoxication far outlasts that observed in the febrile response. Thus the traditional view of the enhanced clearing function of the reticuloendothelial system as the cause of the tolerance1)has been criticized and evidences indicating the stability of carbohydrate metabolism in liver in the tolerant state have been stressed. METHODS Rabbits weighing about 2.5 kg were used. They were maintained with "Okara" (soybean curds waste: 300 g with salt 0.5g daily). For making them tolerant to endotoxin successive increasing doses of endotoxin(salmonella typhosa, Difco)were given intravenously for six days; 10, 20, 40, 100, 200, 200ƒÊg/kg. All the observations of Received for publication June 28, 1963.

2 ENDOTOXIN TOLERANCE animals after test endotoxin administration(300Đg/kg,i.v.)were made in a free state without any restraint.the technique for determinations of the blood sugar and liver glycogen and adrenalectomy were the same as those reported previously2).the thyroid medication and the chemical determinations for dete.cting its effect was reported in a preceding paper3). RESULTS Duration of the tolerant state As has been demonstrated previously4)the susceptibility of rabbits to endotoxin undergoes a marked seasonal variation,being increased in winter and the least in summer.the observations herein reported were concerned exclusively those made during cold season,from late autumn to early spring.even during this season resistance against endotoxin intoxication was found to continue for more than 3 months after cessation of the preliminary endotoxin treatment.the characteristic toxic symptoms to massive doses of endotoxin,such as dyspnea,diarrhea,prostration and hypothermia2) were markedly ameliorated.this long continuance of the tolerance against endotoxin intoxication was quite remarkable in comparison with the ordinary tolerance mainly defined by the febrile response which completely disappears within a period of 2 to 3 weeks1).even in adrenalectomized rabbits made once tolerant the hypothermia and the lethal hypoglycemia were prevented for more than 4 weeks (FIG.1).Presence of adrenal cortex was found to be beneficial "Non -tolerant" "Tolerant" After 1 week After 4 weeks Effect of administration of toxic dose endotoxin(300Đg/kg i.v.)upon rectal temperature and blood sugar level of adrenalectomized rabbits. Resistance to intoxication could be maintained even 4 weeks after cessation of the previous treatment("tolerant").

3 T. FUKUDA AND S. AKIYAMA Liver Glycogen FIG. 2. Prevention of depletion of liver glycogen after toxic dose of endotoxin(300Đg/kg, i. v.)in "tolerant" rabbits. The previous level of liver glycogen were not altered by making the animal tolerant to endotoxin. The tolerant state continued more than 10 weeks after cessation of the previous treatment. in replenishing the liver glycogen depletion after endotoxin which began to reappear after 4 weeks in the absence of adrenal glands. Thus the liver glycogen depletion in intact animals was prevented at least more than 10 weeks after cessation of the previous treatment as shown in FIG. 2. Although the hypertrophy of adrenal cortex was the constant finding in the tolerant state it did not seem to be absolutely necessary for the establishment of the tolerant state. The tolerance here mentioned was found also non-specific and the depletion of liver glycogen after other heterologus endotoxin (E. coli, Difco)was also ameliorated. Mechanism of endotoxin tolerance As has been reported previously2) the depletion of liver glycogen seems to result from the enhanced metabolism in the liver after taking up endotoxin in its reticuloendothelial cells. The preservation of liver glycogen after endotoxin in the tolerant state even in the absence of adrenal cortex might suggest that the enhanced carbohydrate utilization evoked by the uptake of endotoxin might be markedly ameliorated, i. e. the stability of the hepatic carbohydrate metabolism might have been established. In order to test this supposition the effect of administration of thyroid hormone to normal and endotoxin tolerant animals were compared, since it is well documented that thyroid induced acute depletion of liver glycogen5). However, as

4 ENDOTOXIN TOLERANCE Inhibition of thyroid-creatinuria in endotoxin"tolerant"state. this glycogen depleting action of thyroid is not so marked in rabbits as in rats3,6),the appearance of creatinuria related to it was used as a delicate index to the metabolic response to thyroid(fukuda and KOYAMA3)).As shown in FIG.3 the thyroid-creatinuria was found to be difficult to be induced in the tolerant state.in non-tolerant animal even one-half of the dose of thyroid here used could induce a marked creatinuria.as will be discussed later this might also indicate the reduced susceptibility of metabolism especially in the liver. DISCUSSION ABERNATHY et al.7)observing also the long continuance(at least for 10 months) of the resistance to endotoxin in mice following preliminary injection has already suggested that factors other than hyperactivity of the reticuloendothelial system might be important.here it has been demonstrated that one of the basic mechanisms of the tolerance seemed to be the stabilization of the liver metabolism which prevents the depletion of liver glycogen,responsible for the late prostration(fukuda2)).this characteristic feature of the tolerance far outlasted the so-called tolerance observable in the"macromolecular hematic syndromes",such as febrile and leucocytic response,in which the rapid clearing of endotoxin by the reticuloendothelial system might play a dominant role1). Thus in spite of the full recovery of the"macromolecular hematic syndrome" even in response to small doses of endotoxin the tolerant animal did not show

5 T.FUKUDA AND S.AKIYAMA any intoxication syndrome after massive dose of endotoxin at least for 3 months after cessation of the previous endotoxin treatment. How the metabolic stability in the endotoxin tolerance has been induced and what is the characteristic of this state require further clarification.in this respect the fact that the metabolic stress due to thyroid was also reduced in the tolerant state might give some light upon these problems. Concerning the mechanism of inhibition of thyroid-creatinuria in the tolerant state the following may be mentioned.as has been reported in a preceding paper3)the thyroid-creatinuria induced by non-toxic dose of thyroid was found to be due to the manifestation of glucocorticoid action induced by the conditioning action of thyroid.it disappeared after adrenalectomy and reappeared after giving small dose of cortisone which by itself was insufficient to induce creatinuria.sufficient supply of calorie was found also to be effective in preventing the thyroid-creatinuria.it seemed,therefore,to be an accompanying phenomenon of gluconeogenesis due to glucocorticoids for replenishing the liver glycogen,excessively consumed by thyroid action.in the state of tolerance in spite of the marked inhibition of the thyroid-creatinuria no alteration of the cortisone-creatinuria could be observed(fig.omitted.refer3)).thus it might be suggested that the conditioning action of thyroid for the induction of glucocorticoid-creatinuria,perhaps the wastage of liver glycogen,may be reduced in the tolerant state.in this connection it deserves special attention that ascorbic acid which is known to inhibit the thyroid-creatinuria8),but not cortisonecreatinuria9)could also inhibit the depletion of liver glycogen after endotoxin even in adrenalectomized,non-tolerant rabbits(fukuda and KOYAMA10)).Moreover,it was found that during the non-susceptibility to endotoxin of rabbits in hot summer season4)the thyroid-creatinuria was also found difficult to be induced (unpublished data).all these evidences might suggest the intimate relation between the state of tolerance and the resistance to metabolic stress due to thyroid. It has been previously concluded that the intoxication syndrome evoked by endotoxin in rabbits might be induced by the two different mechanisms (FUKUDA2)):one relating to the central autonomic disturbances responsible for the dyspnea and diarrhea and the other relating the peripheral metabolic derangement(the depletion of liver glycogen)responsible for the late prostration and lethal outcome.the present discussion concerned only with the latter mechanism in the tolerant state.how the former mechanism is inhibited remains to be elucidated.ascorbic acid which was effective in preventing the latter mechanism could not inhibit the former mechanism. SUMMARY The resistance against endotoxin intoxication in endotoxin-tolerant rabbits

6 ENDOTOXIN TOLERANCE was found to outlast far the ordinary "tolerance" in relation to the "macromolecular hematic syndrome" in which the clearing of endotoxin by the reticuloendothelial system might play a dominant role. The most characteristic in the tolerant state in relation to the resistance against the lethal effect of endotoxin was the lessening of the liver glycogen depletion after massive dose of endotoxin. That this might be due to the stability of liver metabolism was also suggested by the reduced metabolic response to thyroid in the tolerant state. The creatinuria induced by non-toxic dose of thyroid was found to be inhibited in the tolerant state. REFERENCES 1) BENNET, I. L. AND CLUFF, L. E. Bacterial pyrogen. Pharmacol. Rev. 9: 427, ) FUKUDA, T. On the mechanism of endotoxin intoxication in rabbits. Jap. J. Physiol. 13: 155, ) FUKUDA, T. AND KOYAMA, T. Mechanism of thyroid-creatinuria and its relation to adrenal cortex. Jap. J. Physiol. 13: 479, ) FUKUDA, T. Seasonal variations in endotoxin intoxication of rabbits. Jap. J. Physiol. 13: 240, ) COGGESHALL, H. C. AND GREENE, J. A. The influence of desiccated thyroid gland, thyroxin, and inorganic iodine, upon the storage of glycogen in the liver of the albino rat under controlled conditions. Am. J. Physiol. 105 :103, ) BURN, J. H. AND MARKS, H. P. The relation of the thyroid gland to the action of insulin. J. Physiol. 60: 131, ) ABERNATHY, R. S. AND SPINK, W. W. Protection against brucella with heterologous endotoxin. J. Clin. Invest. 35: 687, ) FISCHER, Gg. UND OEHME, C. Vitamin C and thyreotoxische Kreatinurie. Klin. Wschr. 16: 1453, ) FUKUDA, T. AND KOYAMA, T. Mechanisms of inhibition of thyroid-creatinuria by ascorbic acid. (in preparation) 10) FUKUDA, T. AND KOYAMA, T. Prevention of liver glycogen depletion in endotoxin intoxication by ascorbic acid. Nature. In the press.