Liver Glycogen J. E. Rossouw, Theories

to explain

portacaval failed

to take

rats, followed

compared rats (PFC) equally

of

study

significantly to ALC and

of PCS

ALC

rats.

lower

had markedly plasma

ALC rats, with

liver and

in

with

Plasma plasma

reduced com-

glucose

was

insulin the result

glucagon

concentrations

control

and than

had

glucose

inverse

relationship

glucose

and

was

found,

directly blood

tive

ratios

thus an

between

plasma

I:G

ratio that

was

the

mean for each the

group

blood

primary

experiments

atrophy

related

to

from

after the

gluevent

the

liver,

hypoglycemia, be due

in total and

indicate

PCS appears shunting

is related

Reduction to

I:G

the I :G ratio for each group.

away

appear

com-

concentrations,

controlled

glycogen

intake.

of 1.3:1

intermediate

suggesting

that hepatic

liver

mean

concentration

These

ratio

in the ALC rats. The pair-fed

groups

and plasma

determining

rats)

molar

to 10.2:1

PCS

in PCS rats and PCS rats

elevated

insulin:glucagon

cose

was

Shunt in Rats

and A. S. De Villiers

Liver

control

glycogen PFC

wt

sham-

normal

in all animals S,

rats.

pair-fed

pair-fed

(PCS,

body

reduced rats,

However,

10%

A. I. Vinik.

pared

ad libitum-fed

of ALC

significantly lower

than

gain

reduced to

intake

to a 20%

rats.

pared

effects frequently sequelae

lost

6)

intake

the

In this

also

was

food

account

and

compared

operated

have

up to 42 days postoperatively, food

controls

weight

metabolic

anorexia.

had a lower

D. Labadarios,

(PCS)

into

PCS-induced

(ALC)

the

shunt

After Portacaval

while

of

reduced

to decreased body weight, elevated

to a combination

to be portal

I:G

food relaratios of fac-

tors.

that they had a low

TARZL AND COWORKERS demonstrated, in 1964, that portal diversion reduces liver glycogen in dogs.’ This observation led to the application of the technique to treat human glycogen storage disease, at first by means of portacaval transposition’.3 and later by means of the simpler portacaval shunt procedure.‘.“.” A review of the first 13 patients treated’ indicates that portal diversion for types I, III, and VI, glycogen storage disease usually ameliorated hypoglycemia, acidosis, and hyperlipidaemia. A diminution of liver size, but not necessarily of liver glycogen content, was also observed. Starzl proposed that diversion of portal blood nutrients (mainly glucose) and the pancreatic hormones glucagon and insulin, away from the liver, caused hepatic atrophy and reduced liver glycogen.’ However, the validity of the original hypothesis can be questioned. Portacaval shunting (PCS) and liver injury may, itself, alter glucagon and insulin levelsg*“‘~” and, moreover, reduced food intake following the operation12 may account for some of the observed effects. In fact, PCS dogs survive the operation by no more than a few weeks before dying of inanition and encephalopathy.‘” Similarly, PCS rats lose weight and by 20 wk have not yet regained their preoperative weight.‘” In the present study of PCS rats changes in dietary intake and body weight were followed over a 6-wk period. Appropriate pair- and ad libitum-fed controls

S

From the Department of Medicine, University ofStellenhosch and rksgerherg Hospital. Ihe M R.C‘ National Research lnsritute for Nutritional Diseases. Tygerberg and the Departmen of Medicine. Endocrine and Diabetes Research Group, Universir_vof Cape Town and Groore Schuur HoJpilal. (‘ape Town, Republic ofSouth Africa. Receivedforpublicntion November29. 1977. Supporred bv theSouth African Medical Research Council. ,4ddress reprint requests to J. E. Rossouw, Director, M.R.C. National Research Insrirutefor lutritional diseases, P.O. Box 70, Tygerberg 7505. Republic ofSouth .4frica. c 1978 hy Grune & Strarron. Inc. 00260495/78/2709 -0007%Ol.O0/0 Metabohsm. Vol 27. No 9 (September), 1978

1067

1068

ROSSOUW

ET AL.

allowed an evaluation of the relative importance of PCS-induced changes in dietary intake upon liver weight, liver glycogen content and plasma glucose, insulin and glucagon. MATERIALS

AND METHODS

Male rats (BD9 strain, 230-280g) were housed individually in metabolic cages fited with wide wiremesh bottoms and allowed to adapt to an inverted day-night light cycle (12 hr on; 12 hr off) for a period of I wk prior to experimentation. The portacaval shunt operation (n = 19) was performed under ether anesthesia as described by Lee et aLI5 and the hepatic ischemic time was recorded. Following the operation, the animals were allowed free access to water and food (mashed-chow feed-Epol Limited). The sham operation (n = 14) was performed under similar conditions and the sham operated animals were individually matched in terms of ischaemic time and weight with PCS rats. The mean is ischemic time for both groups was 13 min (range 12-16 min). Both sham-operated (S) and unoperated control (PFC, n = 1I) animals were pairfed with PCS rats. A further group of rats fed ad libitum were included (ad lib controls, ALC, n = 8). Fresh food to all groups was provided in the first half of the night phase of the light cycle. No food was given on the day of sacrifice. All animals were observed for 42 days, from the day of the operation, and food intake and body weight recorded. In the mid-night phase of the 42nd day, blood was drawn under ether anesthesia from the portal vein in the case of all animals in the S, PFC, and ALC groups, and from the abdominal aorta in the case of the PCS group for insulin and glucagon determination. Glucose was determined by the glucose oxidase method (Boehringer) in plasma drawn from the abdominal aorta in all animals. Samples for glucagon determination were taken into heparinized tubes containing 500 IU Trasylol (Aprotinin Bayer)/lml blood. All samples were centrifuged immediately and the plasma deep frozen for assay. lmmunoreactive insulin was determined by a double-antibody method using Amersham kits and a rat insulin standard. lmmunoreactive glucagon was determined as previously describedI using Unger’s 30K antiserum. The liver was excised, blotted, weighed, dropped in liquid N,, and stored at -20” for glycogen determination.17 Values are expressed as mean + I standard error of the mean. Statistical comparisons were made by using the one-tailed Student’s t test.

RESULTS

The food intake of PCS rats was significantly reduced immediately postoperatively (Fig. 1A). On the seventh postoperative day, it was 8.5 + 1 SEM 0.4 g, compared to the preoperative intake of 15.2 f 0.4 g. Although PCS rats increased their food intake thereafter to 11.2 + 0.4 g on the 42nd day postoperatively, their intake was significantly diminished throughout the experimental period when compared to ALC rats. S and PFC rats ate their reduced rations within 4-6 hr, while PCS and ALC rats continued to nibble at their food throughout the 12-hr dark phase and still had food remaining at 24 hr. The body weight of the PCS rats, on the seventh day postoperatively, was 88.3% + 0.6% of their initial weight, and this was significantly lower than that of the sham-operated rats (91.7% =t 1.4%), but not that of pair-fed controls (88.2% i 0.6%, Fig. 1B). The relatively lower body weight of PCS rats was maintained throughout the experimental period and the animals did not regain their preoperative weight. Sham-operated animals attained their preoperative weight, but pairfed controls were only 94% & 1.4% of their weight before the initiation of dietary restriction. Ad lib controls, however, increased their body weight to 125% & 3.1% of their initial weight over the same period. The liver weight of the PCS rats was significantly lower than that of S (p < O.OOOS),PFC (p < 0.0005) and ALC (p < 0.0005) rats (Table 1). Pair-fed animals, both S and PFC, also had significantly lower liver weight than ALC rats (p < 0.025). However, when liver weight was expressed as a function of body

LIVER GLYCOGEN

AFTER PORTCAVAL

SHUNT

1069

IN RATS

ALC

PFC PCS Fig. 1. The food intake in g (A) and percentage loss in body weight (6) in PCS rats and groups of controls.

601 7

14 28 21 DAYS POSTOPERATIVE

35

L2

weight to compensate for differences in the latter parameter, only the PCS rats showed a significant reduction (p < 0.0005) when compared to each of the three control groups. Hepatic glycogen content was significantly reduced, not only in the PCS rats. but also in the S and PFC group when compared to ALC rats (Table I). To compensate for differences in liver and body weight between the groups, hepatic glycogen was also expressed/total liver per 100 g body weight. The reduction in hepatic glycogen of PCS, S and PFC rats when compared to ALC controls remained unchanged. Plasma glucose was significantly lower in the PCS rats when compared to the S (p < 0.025), PFC (p < 0.005) and ALC (p < 0.0005) groups. However, both S and PFC rats also had a significantly (p < 0.025) reduced plasma glucose when compared to the ALC group (Table 1). Table

1.

The

Liver

Weight.

Hepatic

Glycogen, PCS

and

tn = 19)’

Plasma

Glucose

s In =

141

in

PCS, S, PFC, and ALC Rats PFC In =

11)

ALCin

= 81

Lwer Weight (gl

4.01 f 8.6

715+023

6503.019

9.09 :t 0 42

Liver Webght (g/100 g Body Wt)

1 66 f 0.05

281

277

i.006

297

IO09

Hepatlc Glycogen (g/ 100 g Lwer) Hepatvz Glycogen (mg/lOO g

198*023

1 79 * 0 35

130

i 013

640

i- 085

1~37

33.0 f 4 3

Body Wt) Plasma Glucose (mg/lOO n-11 ‘Values are mean i

1 SEM.

108.4*86

iOO6

5191

109

359

1446+

145

1556i

11 9

194i 189

289

t

84

1070

ROSSOUW

ET AL.

600

80 60

PCS

s

PFC

ALC

Fig. 2. The circulating plasma glucagon in pg/ml (A). insulin in plJ/ml (B) and the insulin:glucagon molar ratios (C) in PCS rats and groups of controls.

PCS rats had the highest circulating plasma glucagon levels (679 & 97 pg/ml). These levels just failed to reach statistical significance when compared to the S group (458 & 87), but were significantly higher than those of the PFC (336 & 53, p < 0.025) and ALC (337 * 79, p < 0.025) groups (Fig. 2A). Conversely, PCS rats had the lowest circulating plasma insulin levels (26.3 * 1.4 pU/ml), which were significantly less than S (39.9 & 7.6, p < 0.05), PFC (36.5 f 7.1, p < 0.05) and ALC (91.3 + 11.9, p < 0.0005) groups. Furthermore, plasma insulin was significantly reduced in S and PFC rats when compared to the ALC group (p < 0.005, Fig. 2B). The insulin:glucagon molar ratio (Fig. 2C) was lowest in PCS rats (1.3 f 0.1) and it was significantly different from the S (3.5 + 1.0, p < 0.025), PFC (3.5 f 0.7, p < 0.0025) and ALC (10.2 * 2.8, p < 0.0025) groups. S and PFC rats, in turn, had significantly lower I:G ratios than ALC rats (p < 0.01). There was a significant correlation (r = 0.57, p < 0.001) between plasma glucose and I:G ratio for all rats in the PCS, S, PFC, and ALC groups together. DISCUSSION

Earlier studies on the effects of PCS on body weight, liver weight, and liver glycogen neglected to consider the effects of operation itself and the effects of PCS on food intake with its consequent metabolic sequelae.

LIVER GLYCOGEN

AFTER PORTCAVAL

SHUNT

IN RATS

1071

Certain of the metabolic sequelae of PCS in the rat, such as hepatic atrophy, appear to be directly related to the shunting of portal blood away from the liver, since diminution in liver weight was disproportional to the decrease in body weight. Other effects such as the reduced liver glycogen seem to be related to the PCS-induced reduction in food intake, since hapatic glycogen content was equally reduced in PCS and both pair-fed control groups. However, not all of the effects of PCS were due to a decrease in food intake. Reduction in total body weight, a fall in blood glucose concentration, and elevated insulin-glucagon ratios, although associated with sham operation and with reduced food intake, were exaggerated in PCS. The reduced food intake of PCS rats extended beyond the immediate postoperative catabolic phase: therefore, it is unlikely to be due to the operation per se or liver necrosis. Alterations in serum transaminases are transient following PCS in the rat.” Anorexia may have been due to a minor degree of portosystemic interruption of a portal vein encephalopathy’” or to septicemia. I4 Alternatively, receptor-hypothalamic reflex arc’” may be implicated. Reduction of liver glycogen occurs during fasting” and also after liver injury.” Hormonal mediators of liver glycogen depletion include hypoinsulinemia and increased glucagon and catecholamine concentrations.g’J Although an insulin-glucagon (I:G) ratio of 1.3 + 0.2 was present in the PCS rats, equally severe reduction in liver glycogen in sham and pair-fed control groups in spite of higher, but not normal, I:G ratios (3.5 f 1.0 and 3.5 * 0.7) was found. The reduced circulating insulin levels in the presence of normal, or slightly raised, glucagon levels might be sufficient to enhance glycogenolysis.“’ Alternatively, the altered 1:G ratio may have been the response to decreased food intake,“” with attendant, but not consequent, glycogen depletion. In PCS rats the hepatic arterial route of delivery of hormones may alter their effect on hepatocytes, L’fi offering an explanation for lack of further glycogen depletion in this group. Diversion of “hepatotrophic factors” or reduced hepatic blood flow after PCS did have some specific effect not related to dietary intake, since only PCS rats demonstrated hepatic atrophy disproportionate to the loss of total body weight. In a series of experiments in dogs, Starzl has demonstrated that diversion of “hepatotrophic factors” (gl ucose, insulin, glucagon) is more important than reduced blood flow as a cause of portoprival hepatic atrophy.” An extension of these studies offers fairly conclusive evidence that insulin is the major portal factor responsible for maintaining cell size and number.“: The present findings are in partial agreement with this conclusion, since the PCS rats, the only group with disproportionate liver atrophy, may have had the lowest insulin availability to the liver. The arterial insulin concentration as measured in the PCS group was significantly lower than the portal vein insulin of the control groups; since it is unlikely that blood flow to the PCS liver exceeded that of nonshunted contro1s2* this difference may have been accentuated. Plasma glucose was lower in both pair-fed control groups than in ad lib controls, probably as a result of the enforced reduction in food intake. However, for PCS rats reduced food intake only partly explains their very low plasma glucose compared to ALC rats, since the PCS group also had a lower plasma glucose than both pair-fed control groups. The significance of this observation is enhanced by the change in feeding pattern of the latter, which led to their having a period of fasting prior to sacrifice. Hyperinsulinemia due to impaired hepatic degradation”

1072

ROSSOUW

ET AL.

in PCS rats was not present; in fact, plasma insulin was significantly lower in this group. The plasma insulin:glucagon molar ratio of 1.3 f 0.1 observed in PCS rats would be expected to lead to hyperglycemia; 2gits failure to do so implies ineffective glucagon-mediated glycogenolysis and/or gluconeogenesis. A similar situation pertains in prolonged fasting, where substrate lack has been implicated.30 It is known that although glucagon produces profound and rapid changes in liver metabolism, this effect may be evanescent. :z’Possibly the prolonged hyperglucagonemia associated with PCSg.‘O leads to tachyphylaxis, or the glucagon may be of a relatively ineffective M.W. species. 32The linear relationship between plasma glucose and 1:G ratio for all the rats together suggests that alteration in plasma glucose is the primary event underlying changes in concentrations of insulin and glucagon. In PCS rats there may be a loss of control over glucose homeostasis, as illustrated by a report that PCS rats exhibit relative hypoglycemia at 2 hr and an elevated serum insulin response to an oral glucose load.33 The low I:G ratio in PCS rats is mainly due to increased glucagon. Alterations in hepatic degradation of glucagon34.35 or of insulin3fi may have contributed. Alternatively, overproduction of glucagon, perhaps stimulated by relative hypoglycemia or by hyperaminoacidemia28*37 may have occurred. Hyperglucagonemia has also been described in other catabolic states, such as severe trauma or infection.38.3g Similar changes observed after PCS may, at least in part, be a non-specific response to injury and catabolism. PCS in the rat produces a profound disturbance of glucose-glucagon-insulin homeostasis, which almost certainly has a multifactorial pathogenesis. PCS-induced anorexia, while not accounting for all the changes observed, may account for many of those previously attributed to PCS. In this respect, the marked species variation in the response to PCS should be borne in mind. The dogI and the pig40 show marked anorexia and wasting after PCS: similar changes have not been reported in man. ACKNOWLEDGMENT The technical assistance of P. Child, G. S. Borchardt, and A. B. Kriegler is gratefully acknowledged. Thanks are due to Professor S. J. Saunders, under whose guidance the study was initiated, and to Professor M. A. de Kock for continued support.

REFERENCES I. Sexton AW, Marchioro TL, Waddell WR, et al: Liver deglycogenation after portacaval transposition. Surg Forum 15:120-122, 1964 2. Starzl TE, Marchioro TL, Sexton AW, et al: The effect of portacaval transposition on carbohydrate metabolism: Experimental and clinical observations. Surgery 57:687-697, 1965 3. Riddell AG, Davies RP, Clark AD: Portacaval transposition in the treatment of glycogen-storage disease. Lancet 2: 1146-1148, 1966 4. Hermann RE, Mercer RD: Portacaval shunt in the treatment of glycogen storage disease: Report of a case. Surgery 65:499-503, 1969 5. Boley SJ, Cohen MI, Gliedman ML: Sur-

gical therapy of glycogen rics 46:929-933, 1970

storage

disease.

Pediat-

6. Starzl TE, Brown BI, Blanchard H, et al: Portal diversion in glycogen storage disease. Surgery 65:504-506, 1969 7. Starzl TE, Putnam, CW, Porter KA, et al: Portal diversion for the treatment of glycogen storage disease in humans. Ann Surg 178:525-539, 1973 8. Starzl TE, Francavilla A, Halgrimson CG, et al: The origin hormonal nature and action of portal venous hepatotrophic substances in portal venous blood. Surg Gynecol Obstet 137:179-199, 1973 9. Sherwin

R. Joshi

P,

Hendler

R,

et

al:

LIVER GLYCOGEN

AFTER PORTCAVAL

SHUNT

IN RATS

Hyperglucagonaemia in Laennec’s cirrhosis. N Engl J Med 290:239-242, 1974 IO. Shurberg JL, Resnick RH, Kott RS, et al: Serum lipids, insulin, and giucagon after portacavai shunt in cirrhosis. Gastroenteroiogy 72:3Oii304. 1977 I I. Bucher NL, Weir CC: Insulin, giucagon. liver regeneration, and DNA synthesis. Metabolism 25:1423-1425. 1976 12. Kvu MH, Cavanagh JB: Some effects of portacaval anastomosis in the male rat. Brit J ExpPath51:217 227, 1970 13. Fischer JE, Funovics M, Aguirre A, et al: The role of plasma amino acids in hepatic encephaiopathy. Surgery 78:276- 290, 1975 14. Keraan M, Meyers OL. Engeibrecht GHC, et al: Increased serum immuno globulin levels following portacavai shunt in the normal rat. Gut I5:468 -472, 1974 15. Lee S, Arnot RS, Engelbrecht GCH, et al: Microvascular surgery in South Africa: Part II operative techniques. S Afr Med J 47:1596 -1600, 1973 16. Vinik Al, Hardcastle A: Structure antigenicity relationships of glucagon and related peptides. Horm Metab Res 6:3955399, 1974 17. Seifter S, Dayton S, Novic B, et al: Estimation of giycogen with anthrone reagent. Arch Biochem 25:191 200, 1950 IX. Meyers OL. Hickman R, Keraan M, et al: .4cute biochemical and histological effects of portacaval shunt in the normal rat. S Afr Med J 49:1048 1050, 1975 19. Baldessarini RJ, Fischer JE: Serotonin metabolism in rat brain after surgical diversion of the portal venous circulation. Nature [New Biol] 245125-27. 1973 20. Schmitt M: Influences of hepatic portal receptors on hypothalamic feeding and satiety centers. Am J Physiol225:10891095, 1973 21. Huitman E. Nilsson LH: Liver glycogen in man. Effect of different diets and muscular exercise, in Pernow B, Saitin B (eds): Muscle Metabolism During Exercise. New York, Plenum Press, 1971, pp 143. I5 I 22. Felig P, Brown WV, Levine RA, et al: Glucose homeostasis in viral hepatitis. N Engi J Med 283:1436 1440, 1970 23. Levine R. Haft DE: Carbohydrate stasis. N Engi J Med 283:175-183. 1970

homeo-

24. Unger RH. Aydin I. Nakabayashi H. et al: The effects of giucagon administration to nondiabetics and diabetics. Metabolism 25:1523 1526, 1976 25. Gerich J. Charles M, Grodsky G: Regula-

1073

tion of pancreatic insulin and giucagon secretion. Annu Rev Physiol38:353-388, 1976 26. Price JB: Insuhn andglucagon as moditirrs of DNA synthesis in the regenerating rat iner. Metabolism 25: 1427 1428, 1976 27. Starzi TE, Porter KA, Putnam CW: Insulin. glucagon. and the control of hepatic structure, function, and capacity for regeneration. Metabolism 25:1429--1434, 1976 28. Redeker AC, Gelier HM. Reynolds TB: Hepatic wedge pressure, blood Row, vascular resistance and oxygen consumption in cirrhosis before and after end-to-side portacaval shunt. .i Ciin invest 37:606-618, 1958 29. Cherrington AD, Chiasson JL, Liijenquist JE, et al: The role of insulin and glucagon in the regulation of basal glucose production in the portabsorptive dog. J Ciin Invest 58:1407 1418. 1976 30. Feiig P: The glucose-aianine cycle. Metabolism 22:179--207. 1973 3 I. Sherwin R. Wahren J, Felig P: Evanescent effects of hypo- and hyperglucagonaemia on blood glucose homeostasis. Metabolism 25:138i- 1383, 1976 32. Valverde I, Vilianueva ML: Hetrrogeneity of piasma immunoreactive glucagon. Metabolism 25:1393-1395. 1976 33. Assal JP, Levrat R, Stauffecher W. et al: Metabolic consequences of portacaval shuntmg in the rat: effects on glucose tolerance and serum immunoreactive insulin response. Metabolism 20:850- 858, 1971 34. Assan R: In viva metabolism of glucagon. in Lefebvre PJ, Linger RH (eds): Glucagon. Oxford, Pergamon Press. 1972. pp 47 59 35. Jaspan JB. Rubenstein AH: Plasma profiles and metabolism in health and disease. Drabetes 26:887 -902, 1977 36. Blackard WC, Nelson NC: Portal and pe ripheral vein immunoreactive insulin concentrations before and after glucose infusion. Diabetes 19:302-306. 1970 37. James JH, Hodgeman JM, Funovics JM. et al: Brain tryptophan, plasma free tryptophan and distribution of plasma neutral amino acids. Metabolism 25:47 i-476. 1976 38. Bloom S: Glucagon, a stress hormone. Postgrad Med J 49 (August Suppi): 607 61 I, I973 39. Unger R: Giucagon and the insulin: Giucagon molar ratio in diabetes and other catabolic diseases. Diabetes 20:834- 838. 197 I 40. Hickman R, Terbianche J: Anorexia and weight loss in the portacavai-shunted pig. Surgery 80:569 574, 1976

Liver glycogen after portacaval shunt in rats.

Liver Glycogen J. E. Rossouw, Theories to explain portacaval failed to take rats, followed compared rats (PFC) equally of study significantly...
570KB Sizes 0 Downloads 0 Views