Nutrient Requirements

and Interactions

HARIHARAN SANKARAN,3 CLIFFORD W. DEVENEY, EDWARD C. LARKIN* AND G. ANAHDA RAO* Surgical Service, VA Medical Center, Portland, OR 97207 and Department of Surgery, Oregon Health Sciences university, Portland, OR 97201 and *Alcohol Research Laboratory, VA Medical Center, Martinez, CA 94553 (Lieber and DeCarli, 1989). Several studies have shown that pancreatic amylase activity is reduced in rats fed a 36% alcohol diet, and the reduction was attributed to alcohol per se (La Sure et al. 1986, Ponnappa et al. 1986; Schmidt and Pandol, 1990) or to the differential effects of alcohol on the pancreas, depending upon the carbohydrate concentration in the alcohol diet (Ponnappa et al. 1986). This reduction in amylase activity, however, may be due to insuffi cient ingestion of carbohydrate, because rats fed the 36% alcohol diet have been shown to ingest small amounts of carbohydrate (Rao et al. 1987, Rao and Larkin 1984). Furthermore, changes in liver histology caused by chronic alcohol ingestion can be avoided by altering various nutritional factors in rats (Bosma et al. 1991, Nanji et al. 1989, Porta et al. 1972, Rao et al. 1986 and 1987, Rao and Larkin 1984, Yonekura et al. 1989) and baboons (Ainley et al. 1988, Lieber et al. 1990) even in the presence of high BAL. It is likely that the decrease in pancreatic amylase activity also can be alleviated despite BAL if rats ingest substan tially more carbohydrate. For this purpose, we ex amined the effect of a daily intake of alcohol adequate to cause alcoholemia on pancreatic enzyme activity in rats ingesting different amounts of carbohydrate. In addition, we studied cholecystokinin-octapeptide-

ABSTRACT Adverse effects observed in alcoholic rats are often attributed to alcohol per se. Alcoholic liver damage, however, can be avoided by modulating nutri tional factors despite high blood alcohol concentrations. Hence, we examined the effect of blood alcohol concen tration on pancreatic enzyme activity and release. Three liquid diets containing 36 and 18% of total energy derived from alcohol and protein, respectively, were fed. Each alcohol diet contained 11, 21 or 31% of energy from carbohydrate, and the fat concentration was ap propriately adjusted. The control groups of rats (fed an isoenergetic liquid diet without alcohol) and the alcoholic groups of rats were maintained for 2 wk. The three groups of alcoholic rats consumed 13.3 ±2.3, 13.3 ± 2.2 and 13.2 ±1.9 g/kg of alcohol daily, and their corresponding blood alcohol levels were 41.5 ±4.3, 55.4 • 8.9 and 44.6 ±2.2 mmol/L. Pancreatic acinar amylase activity in alcoholic rats was proportional to carbohy drate ingested, despite high blood alcohol concentra tions; chymotrypsin and trypsin activities were un changed. Acinar enzyme activities in control rats were similar. Furthermore, cholecystokinin-octapeptidestimulated amylase release in alcoholic rats corre sponded with the amylase concentration in acini, whereas stimulated trypsin output was unaltered in both control and alcoholic rats. These results demonstrate that neither alcohol ingestion nor high blood alcohol concentration affects the activities of pancreatic pro teases and that the changes in the activity and release of amylase are related to the intake of carbohydrate. J. Nutr. 122: 1884-1891, 1992. INDEXING KEY WORDS:

•alcohol •carbohydrate intake •rats •pancreatic acini •amylase activity

Organ damage in chronic alcoholism is considered to result from a condition in which alcohol intake is associated with high blood alcohol levels (BAL) 0022-3166/92

$3.00 ©1992 American

Institute

of Nutrition.

'Data presented in part at the American Pancreatic Association meeting, October 31-November 1, 1991, Chicago, IL (Sankaran, H., Deveney, C. W., Larkin, E. C. & Rao, G. A. (1991| Pancreatic enzyme content and secretion in chronic alcoholic rats: insig nificant role for high blood alcohol levels. Pancreas 6: 718 (abs.)]. Supported by the Department of Veterans Affairs Medical Re search Service. •'"To whom correspondence should be addressed at Surgical Service (112P), VA Medical Center, P.O. Box 1034, Portland, OR 97207.

Received 11 December

1884

1991. Accepted 29 April 1992.

Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1884/4769466 by Tulane University Medical Library user on 19 January 2019

Carbohydrate Intake Determines Pancreatic Acinar Amylase Activity and Release despite Chronic Alcoholemia in Rats1'2

BLOOD ALCOHOL

AND PANCREATIC

ENZYMES

1885

TABLE 1 Composition of liquid diets used in the study* Low carbohydrate-high fat diet Nutrient

Control

Alcohol

Med carbohydrate-med fat diet Control

Alcohol

High carbohydrate-low fat diet Control

Alcohol

(36) Carbohydrate (maltose-dextrin)Protein CaseinL-Cystine DL-MethionineFat Corn oilOlive oilSafflower oilFiberSodium

(47)42.2 (18) 41.40.5

(11)42.2 25.6 (18) 41.40.5

0.339.6 0.339.6 (35) (35) 8.528.42.710.02.0 8.528.42.710.02.0

(36) (57)42.2 (18) 41.40.5

(21)42.2 50.6 (18) 41.40.5

0328.3 0.328.3 (25) (25) 6.120.31.910.02.0 6.120.31.910.02.0

(36) 165.7 (67)42.2 (18) 41.40.5

(31)42.2 77.2 (18) 41.40.5

0.317.0 0.317.0 (15) (15) 3.612.21.210.02.0 3.612.21.210.02.0

carrageenate Vitamin mix Salt mix20.0115.2

2.5 2.5 2.5 2.5 2.5 2.5 8.7550.0 8.750.0141.9 8.7550.0 8.750.0 8.7550.0 8.75 'Values in parentheses represent energy contribution as a percentage of total calculated on the basis of KJ/g of nutrient: ethanol 29.7, protein 17.9, carbohydrate 16.6, and fat 37.0. One mL of diet provides 4.184 KJ. Compositions of vitamins and salts are as recommended by the American Institute of Nutrition (AIN 1977).Vitamin concentrations per liter of liquid diet are as follows: thiamine HC1, 1.5 mg; riboflavin, 1.5 mg; pyridoxine HC1, 1.75 mg; nicotinic acid, 7.5 mg; Ca pantothenate, 4 mg; folie acid, 0.5 mg; biotin, 0.05 mg; vitamin B-12, 25 ng; retinyl palmitate, 12 mg; cholecalciferol, 1 mg; all-rac-ot-tocopherol, 60 mg; vitamin K, 25 ug; PABA, 12.5 mg; inositol, 25 mg; choline, 250 mg. Concentrations of salts are the same as described by AIN (1977)except for the addition of fluoride (0.25 mg/L).

stimulated amylase and trypsin release from acini to determine whether the integrity of cholecystokinin receptors is affected by chronic alcoholemia.

MATERIALS AND METHODS The study was approved by the institutional sub committees for the use of animals at the VA Medical Centers, Portland, OR and Martinez, CA. Male Sprague-Dawley rats weighing -80 g were obtained from Bantin Si Kingman (Fremont, CA) and fed a nonpurified pellet diet (Rodent Lab Chow 5001, Purina Mills, IN) for 3 d. They were then housed individually in metabolic-type stainless steel cages with wire bottoms and weaned to appropriate alcohol liquid diets (Table 1). The liquid diets were fed through Richter-type feeding tubes. The weaning pro cedure was as follows: rats were fed the control liquid diet (without alcohol) with free access for 2 d. Al cohol-fed groups of rats were allowed free access to liquid diets with 12% of total energy as alcohol for 2 d and subsequently with a 24% alcohol diet for 2 d. Rats were then fed the 36% alcohol diet for 2 wk. The 12% alcohol diet was prepared by mixing 2/3 amounts of control diet with 1/3 amounts of 36% alcohol diet, and the 24% alcohol diet was prepared

by mixing 1/3 amounts of control diet with 2/3 amounts of 36% alcohol diet. The control groups of rats were either fed isoenergetic amounts of control diet or were allowed free access to the control diet for 14 d. The isoenergetic amount was determined from the alcohol diet consumed by the alcohol-fed group of rats on the previous day. Rats were fed between 0900 and 1100 h every day, and daily diet intake and weekly weight gains were recorded. The study was performed in two experiments. In Experiment 1, two groups of rats (total of 20) were used. A group of 15 rats were fed the low carbo hydrate-high fat alcohol diet (Lieber-DeCarli 36% al cohol diet; Lieber et al. 1989) (Table 1). Rats were allowed free access to the diet for 14 d. Three rats were anesthetized with a cocktail containing ketamine, acepromazine and xylazine to obtain blood and pancreas after d 1, 3, 5, 7 and 14. Blood was collected by cardiac puncture in a heparinized tube. Plasma was separated by centrifugation at 2500 x g for 10 min and stored in a tightly capped microfuge tube at 4°Cuntil the following morning, when alcohol determination was conducted. The pancreas was re moved and a piece of tissue (-100 mg) was weighed and homogenized in 30 volumes of 50 mmol/L phos phate buffer, pH 7.4, using a Brinkmann polytron homogenizer. The homogenate was centrifuged at

Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1884/4769466 by Tulane University Medical Library user on 19 January 2019

8/LAlcohol

1886

SANKARAN ET AL. 4000 x g for 10 min at 4°Cto obtain a clear super

acteristics of acini isolated from alcoholic and control rats. Amylase (EC 3.2.1.1) activity was determined by the method of Jung (1980) using procion yellow dye coupled to starch as the substrate. Chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) were assayed by the methods of Huttunen (1973) and Hummel (1959), re-

TABLE 2 Pancreatic amylase activity in rats fed the 36% of energy as alcohol diet containing 11% as carbohydrate and 35% as fat1

Pancreatic amylase activity Duration (d|

Tissue

868*2232 ± 2482852 ± 3722108 ± 248992 ± 372+8308 ± (Isoener-geticcontrol]U/g7688

13571414

Acini ±2.1*10.0 1.7*7.0 ± 0.46.7 ± 1.66.2 ± 1.717.4 ±

BAL 6.153.0 ± 5.470.0 ± 0.964.6 ± 10.963.0 ± 3.93.5 ±

±1364*U/mg28.6±3.8*mmol/L53.9 ± 0.2 'Values are means + so of 3-5 rats in each group. Amylase activity is expressed in units/g pancreas (tissue) or U/mg acinar protein (acini). 'Significantly different from the values at 3, 5, 7 and 14 d for rats fed the alcohol diet. +Significantly different from the values at 1, 3, 5 and 7 d (for tissue] and 1, 5, 7 and 14 d (for acini) for rats fed the alcohol diet (P < 0.05).

spectively. Pancreatic acinar protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. Alcohol concen tration in the plasma (BAL)was determined by using alcohol dehydrogenase (EC 1.1.1.1) with a kit ob tained from Sigma Chemical (St. Louis, MO). Richtertype feeding tubes, alcohol and control liquid diets were purchased from BioServ (Frenchtown, NJ). Collagenase was obtained from Worthington Biochemical (Freehold, NJ) and procion yellow from Polysciences (Warrington, PA). Cholecystokinin-octapeptide was obtained from Peninsula Laboratories (Belmont, CA). Hyaluronidase and all other chemicals were obtained from Sigma Chemical. Statistical analysis. One-way ANOVA was used to compare data from various groups in both experi ments. The significance (P < 0.05) between means of specific groups was determined by Bonferroni i test. RESULTS Experiment 1 Rats fed the 36% alcohol diet for 1 d exhibited high BAL, and this level remained high throughout the 14 d. Alcohol ingestion for 1 d did not significantly reduce pancreatic amylase activity in these rats com pared with the control rats (Table 2). Amylase activity of isoenergetic controls was significantly higher when compared with that of alcoholic rats from d 3 through 14. Pancreatic tissue and acinar amylase activity in rats fed the 36% alcohol diet for subsequent days

Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1884/4769466 by Tulane University Medical Library user on 19 January 2019

natant that was used to assay for tissue amylase activity. The remainder of the pancreas (~1 g) was used to isolate acini on the same day for the determi nation of acinar enzyme activity. The second group of five rats was fed the control diet in amounts isoenergetic to those consumed by the alcohol-fed group. After 14 d, they underwent the same experimental protocol as the alcohol-fed group. In Experiment 2, three alcohol liquid diets and their corresponding control liquid diets were used (Table 1). Energy derived from protein and alcohol was maintained constant at 18 and 36% of total, respectively. The carbohydrate and fat concentrations were manipulated appropriately to make the diets isoenergetic. The energy content of the low carbohy drate-high fat alcohol diet consisted of 11 % carbohy drate and 35% fat; the medium carbohydrate-medium fat diet, 21% carbohydrate and 25% fat; and the high carbohydrate-low fat diet, 31% carbohydrate and 15% fat. In the corresponding control diets, energy from alcohol was replaced by carbohydrate. Nine groups of five rats each were used in this experiment. Three groups of five rats each were fed the three alcohol diets with free access to the diet. For each alcohol diet group, two control groups of five rats each were fed the corresponding nonalcoholic liquid diet; one group was fed an amount isoenergetic to that consumed by the alcohol-fed group (isoenergetic control), and the other had free access to the diet (free access control). At the end of 14 d, all rats were killed under anes thesia to obtain pancreas for preparation of acini and blood for determination of BAL, adopting the same experimental protocol used in Experiment 1. Isolation of acini, determination of activities of pancreatic en zymes, and determination of the response of acini to stimulation by cholecystokinin-octapeptide were per formed on the same day. Preparation of acini and amylase release. Pan creatic acini were isolated by a modified procedure as described by Sankaran et al. (1979). Briefly, the pro cedure involves digestion of the pancreas by collagenase and hyaluronidase, followed in succession by mechanical disruption of the tissue and centrifugation through 40 g/L bovine serum albumin. The secretory function of acini was evaluated by stimulating them with varying concentrations of cholecystokinin-oc tapeptide as previously described (Sankaran et al. 1979). Amylase, chymotrypsin and trypsin released into the medium at the end of 30 min of incubation at 37"C were determined to quantify the secretory char

BLOOD ALCOHOL

AND PANCREATIC

1887

ENZYMES

TABLE 3 Effect of feeding liquid diets of different carbohydrate and fat concentrations with (36% of energy) and without of energy, alcohol and macronutrients1

alcohol on intake

kj/kgAlcohol,

g/kgCarbohydrate,

g/kgProtein,

Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1884/4769466 by Tulane University Medical Library user on 19 January 2019

control1072.41064.81154.830.436.646.711.010.911.810.17.24.7±± alcohol1117.01102.11097.013.313.313.27. control2054.31817.11775.758.362.571.221.018.618.219.412.37.2±±±±±±±±±±±Â

Daily intakeEnergy,

*+2.9*+1.9"1.8-1.6*1.8*1

g/kgFat,

g/kgDietABCABCABCABCABCFree-access

'Values are means ±so of five rats in each diet group. Diets: A - low carbohydrate-high fat; B - medium carbohydrate-medium fat diet; C high carbohydrate-low fat diet. 'Significantly different from the values of corresponding free-access control values. ^Significantly different from the values of corresponding

isoenergetic

control group (P < 0.05).

were significantly lower when compared with the values at d 1. In the alcohol diet-fed group, tissue amylase activity on d 14 was significantly lower than on previous days (Table 2). Experiment 2 Nutrient, alcohol intake and blood alcohol levels. Rats with free access to the control diets consumed significantly more macro- and micronutrients when

compared with either the isoenergetic controls or the alcoholic rats because inclusion of 36% of energy as alcohol in the diet caused anorexia (Table 3). The isoenergetic controls consumed, however, more car bohydrate than did the alcoholic rats (Table 3). Daily energy intake in rats with free access to the three alcohol diets was similar, however, despite the dif ferent proportions of carbohydrate and fat in the diets (Table 3). Hence, they ingested similar amounts of alcohol daily when alcohol-derived energy is ex pressed either per rat or per kilogram of body weight

TABLE 4 Effect of feeding control and alcohol (36% of energy) diets with different carbohydrate on pancreatic acinar enzyme activities1 Diet and feeding regimen Low carbohydrate-high fatMedium

BAL accessIsoenergeticFree accessFree

carbohydrate-medium fatHigh

accessIsoenergeticFree accessFree

carbohydrate-low fatFree

accessIsoenergeticFree

Amylase

and fat concentrations

Chymotrypsin

Trypsin

5.335.0 ± 6.05.0 ± 0.4*+29.9 ± 4.3——55.4 ±

0.21.8 ± 0.31.3 ± 0.41.5 ±

0.41.7 ± 0.21.2 ± 0.11.7 ±

2.134.8 ± 1.712.6 ± +36.0 ±1.1* 8.9——44.6 ±

0.21.4 ± 0.31.6 ± 0.21.2 ±

0.41.6 ± 0.21.9 ± 0.11.5 ±

9.240.8 ± 0.31.2 ± 0.31.5 ± 4.321.0 ± 0.31.1 ± 0.41.8 ± ±1.0*+U/mg1.1 accessControlControlAlcoholControlControlAlcoholControlControlAlcoholmmol/L——41.5 ±2.232.7 ±0.31.4 ±0.1 'Values are means ±so of five rats in each group. Enzyme activities are expressed as U/mg acinar protein concentration. 'Significantly different from the free-access and isoenergetic control values within each diet group. ^Significantly different from each other (P < 0.05).

1888 0.4

SANKARAN

ET AL.

—•—Control (47% CHO - 35% Fat)

Alcohol Diet

••••OAlcohol (11% CHO - 35% Fat)

"O—

6.20

11% CHO-35%

Fat

—O— 21% CHO-25%

Fat

•-O-

Fat

31% CHO-15%

—A— Control 47% CHO - 35% Fat

O>

ai

2.48

o.o

0.01

0.1

10

CCK8 (nmol/L)

FIGURE 1 Effect of cholecystokinin-octapeptide (CCK8) stimulation on trypsin output from acini isolated from control and alcoholic rats. Points are the means ±SD of triplicate experiments from four rats for each condition (n 4).

(Table 3). The synergistic effect of the intake of in sufficient macronutrients and high amounts of al cohol resulted in high BAL (Table 4). Values of BAL of 21.7-32.6 mmol/L are not uncommon in rats fed the 36% alcohol diet (Lieber et al. 1989); however, values ranging from 35 to 65 mmol/L have also been re ported (Rao et al. 1988, Sankaran et al. 1989a). In this study, BAL in the three alcoholic groups of rats ranged from 41.5 to 55.4 mmol/L, supporting previous observations (Table 4). Furthermore, the three groups of alcoholic rats ingested significantly different amounts of carbohydrate and fat daily, although their protein intakes were similar (Table 3). Pancreatic chymotrypsia and trypsin. Acinar chymotrypsin and trypsin activities were essentially the same in all nine groups of rats (Table 4). Neither alcohol intake nor high BAL had a significant effect on these two proteolytic enzymes. Even a twofold higher ingestion of protein in free-access control rats did not significantly alter the activities of these two proteolytic enzymes when compared with those of the corresponding isoenergetic or alcoholic groups. Pancreatic amylase. Amylase activity (U/mg acinar protein) was the lowest in rats ingesting the low carbohydrate-high fat alcohol diet (5.0 ±0.4) and markedly higher in those ingesting high carbohydratelow fat alcohol diet (21.0 ±1.0). Rats consuming the medium carbohydrate-medium fat alcohol diet ex hibited intermediate amylase activity (12.6 ±1.1) (Table 4). Amylase activities in the three alcohol diet groups were significantly different from one another (P < 0.05, Table 4). Amylase activities in rats from both control groups were similar and significantly higher than those of the corresponding alcohol-fed groups (Table 4).

''•

I

..4

T

1.24

-o

0.00 0.01

0.1

1

10

CCK8 (nmol/L)

FIGURE 2 Effect of cholecystokinin-octapeptide (CCK8) stimulation on amylase release from acini isolated from control and alcoholic rats. Points are the means ±SD of triplicate experiments from four rats for each condition (n =• 4).

Stimulated enzyme output from acini of chronic alcoholic rats. Output of trypsin and amylase from acini of alcoholic and control rats in response to stimulation with increasing concentrations of cholecystokinin-octapeptide were compared (Fig. 1 and 2). Cholecystokinin-octapeptide-stimulated trypsin secretion was not altered whether the rats ingested the low carbohydrate, high fat alcohol diet or the control diet without alcohol (Fig. 1). Stimulated amylase release was dependent upon the carbohydrate concentration in the alcohol diet (Fig. 2). Response to cholecystokinin-octapeptide of acini from rats fed the low carbohydrate-high fat alcohol diet was poor when compared with that of acini from rats ingesting control diet. Furthermore, an increment in carbohy drate ingestion in alcoholic rats significantly en hanced the amylase release in response to cholecystokinin-octapeptide (Fig. 2).

DISCUSSION Pancreatic enzyme synthesis is regulated in a di rectly proportional manner by the quantity of dietary substrates ingested (Howard and Yudkin 1963, Schick et al. 1984, Wicker and Pugserver 1987). A major determinant is the macronutrient composition of the diet (Howard and Yudkin 1963, Snook 1971), as well as the condition of starvation and refeeding (Danielsson et al. 1974, Lee et al. 1982). Thus, high carbohydrate diets increase pancreatic amylase ac-

Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1884/4769466 by Tulane University Medical Library user on 19 January 2019

3.72

BLOOD ALCOHOL

AND PANCREATIC

1889

the Rao-Larkin 26% alcohol diet, we previously re ported that the low carbohydrate intake and not al cohol consumption per se is the primary cause of pancreatic amylase insufficiency in chronically alco holic rats (Sankaran et al. 1989b). In another study, we showed that partial supplementation with in tramuscular injections of glucose in rats fed the 36% alcohol diet not only significantly reduced BAL but also enhanced pancreatic amylase activity (Sankaran et al. 1989a). However, it was not clear from earlier studies whether the increment in amylase activity was due to an increase in carbohydrate intake or to a reduction in BAL or both. Chronic alcohol intake may affect the pancreas in a number of ways, such as interfering with the ab sorption of nutrients and/or by induction of high BAL. Chronic alcohol intake does not seem to affect the absorption of nutrients regulating pancreatic enzyme activities, because rats fed the isoenergetic 26% al cohol diet consume amounts of alcohol similar to those consumed by rats fed the 36% alcohol diet, and yet do not show significant alteration in pancreatic amylase activity when compared with their isoener getic controls (Sankaran et al. 1989b). If alcohol intake were to interfere with the absorption of nutrients, such malabsorption would happen not only with carbohydrate but also with protein, causing protein malnutrition and potentially a reduction in pancreatic proteolytic enzymes. In rats fed the 36% alcohol diet, however, not all the pancreatic enzymes are altered. These observations in combination with the fact that the pancreas does not metabolize alcohol to a significant degree (Krebs et al. 1969) suggest that the putative effects of alcohol ingestion on the pan creas are mediated by circulating alcohol and/or its metabolite, acetaldehyde. Our data show that, despite chronic alcoholemia, amylase activity in the pancreas is proportional to carbohydrate ingestion, and activ ities of trypsin and chymotrypsin are not altered. These data suggest that neither alcohol ingestion nor high BAL have a dramatic role in adversely affecting pancreatic amylase or other pancreatic enzymes. Fur thermore, carbohydrate intake regulates amylase ac tivity in chronic alcoholic rats in a manner similar to the regulation of amylase synthesis by carbohydrate intake in nonalcoholic rats (Schick et al. 1984, Snook 1971). Amylase activities in both groups of control rats ingesting different proportions of carbohydrate are essentially unchanged, suggesting a requirement for optimum carbohydrate intake for maximal levels of amylase synthesis. Such a regulatory mechanism may also be in operation for the synthesis of chymotrypsin and trypsin, because these enzymes were not altered in any of the rats in spite of ingestion of significantly different amounts of protein. Circulating alcohol has been suggested to alter cel lular membrane integrity (Rubin and Rottenberg, 1982). In the present study, release of trypsin in re sponse to cholecystokinin-octapeptide stimulation

Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1884/4769466 by Tulane University Medical Library user on 19 January 2019

tivity (Snook 1971), high protein diets induce proteolytic enzymes (Snook 1971) and high fat diets enhance lipase activity (Beaudoin et al. 1989, Saab et al. 1986, Wicker and Pugserver, 1987). Furthermore, Schick et al. (1984) demonstrated that dietary increases in pro teins with corresponding decreases in carbohydrate result in a parallel and dramatic decrease in the syn thesis of both forms of amylase. Rats ingesting similar amounts of carbohydrate daily, therefore, exhibit similar amylase activity. The 36% alcohol liquid diet contained only 11% of total energy as carbohydrate, so as to include 36% of energy as alcohol compared with the control diet, which contained 47% of total energy as carbohydrate. This discrepancy in dietary carbohydrate concen tration resulted in the ingestion of significantly lower amounts of carbohydrate by rats fed the 36% alcohol diet compared with rats consuming either the isoenergetic or free-access control diets. Furthermore, rats fed the 36% alcohol diet ingest -50% less food compared with rats given free access to the control diet (Rao et al. 1987, Rao and Larkin 1984, Table 3). Hence, rats fed the 36% alcohol diet consumed signif icantly less carbohydrate when compared with the free-access controls. The isoenergetic control rats also received a higher proportion of carbohydrate when compared with rats fed the alcohol diet. Because many of the previous studies did not consider this low carbohydrate concentration in the diet and the insufficient intake of carbohydrate by alcoholic rats as potential factors in affecting amylase activity (La Sure et al. 1986, Ponnappa et al. 1986, Schmidt and Pandol 1990), alcohol ingestion per se was implicated as the cause for the reduction in pancreatic amylase activity. If alcohol per se were toxic to the pancreas, such a toxic effect should not only cause a reduction in all the enzymes in the pancreas of chronically alcoholic rats, but also be independent of dietary macronutrient composition or concentrations. However, this is not the case, because in most of the studies employing the 36% alcohol diet, the pancreatic activity of amylase alone is reduced (La Sure et al. 1986, Pon nappa et al. 1986, Schmidt and Pandol, 1990). Fur thermore, changes in dietary fat concentration have been shown to proportionately modify lipase activ ities in the pancreas despite ingestion of high amounts of alcohol (Ponnappa et al. 1986). Our earlier studies (Sankaran et al. 1989a and 1989b, Tsukamoto et al. 1985), suggested that the changes in pancreatic amylase activity of chronically alcoholic rats are due to the ingestion of significantly lower amounts of carbohydrate rather than to the adverse effect of al cohol. Tsukamoto et al. (1985) attributed the differ ences in the synthetic and secretory patterns of amylase from the pancreatic acini of rats fed an al cohol diet to both alcohol ingestion and a con comitant reduction in carbohydrate in the diet. Using

ENZYMES

1890

SANKARAN

ACKNOWLEDGMENTS We thank Thong Nguyen for his technical assis tance. We also thank Roberta Ruimy and Kirstin Milne for assistance in the preparation of the manu script.

LITERATURE CITED Ainley, C. C, Senapati, A., Brown, I.M.H., lies, C. A., Slavin, B. M., Mitchell, W. D., Davies, D. R., Keeling, P.W.N. & Thompson, R.P.H. (19881 Is alcohol hepatotoxic in the baboon? J. Hepatol. 7: 85-92. American Institute of Nutrition (1977| Report of the American Institute of Nutrition ad hoc committee on standards for nutri tional studies. I. Nutr. 107: 1340-1348. Beaudoin, A. R., Bigin, M. E., Ells, G., St. Jean, P., Laforest, L., Proulx, J. & Vachereau, A. (1989| Type of dietary lipids exerts a major influence on the secretory activity of the exocrine pan creas: medium-term studies. Pancreas 4: 418-422. Bosma, A., Seifen, W. F., Wilson, J.H.P., Roholl, P.J.M., Brouwer, A. &. Knook, D. C. (1991| Chronic administration of ethanol with high vitamin A supplementation in a liquid diet to rats does not cause liver fibrosis: 1. morphological observation. J. Hepatol. 13: 240-248. Danielsson, A., Marklund, S. & Stigbrand, J. (1974) Effects of star vation and islet hormones on the synthesis of amylase in iso lated exocrine pancreas of the mouse. Acta Hepato-Gastroenterol. 21: 189-197. Howard, F. & Yudkin, I. (1963] Effect of dietary change upon the amylase and trypsin activities of the rat pancreas. Brit. J. Nutr. 17: 281-293. Hummel, B.C.W. (1959| A modified spectrophotometric determi

nation of chymotrypsin, trypsin and thrombin. Can. J. Biochem. Physiol. 37: 1393-1399. Huttunen, R. (1973) Proteolytic enzymes in experimental rat pan creatitis. Ann. Med. Exp. Biol. Fenn. 51: 133-138. Jung, D. H. (1980) Preparation and application of procion yellow starch for amylase assay. Clin. Chim. Acta 100: 7-11. Krebs, H. A., Freedland, R. A., Hems, R. & Stubbs, M. (1969) Inhibition of hepatic gluconeogenesis by ethanol. Biochem. J. 112: 117-124. La Sure, M. M., Jimenez, F. P. & Singh, M. (1986) Interaction of chronic alcohol administration and diet on pancreatic acinar cell metabolism in the rat. Dig. Dis. Sci. 31: 1073-1080. Lee, P. C., Brooks, S. & Lebenthal, E. (1982) Effect of fasting and refeeding on pancreatic enzymes and secretagogue respon siveness in rats. Am. J. Physiol. 242: G215-G221. Lieber, C. S. & DeCarli, L. M. (1989) Recommended amounts of nutrients do not abate the toxic effects of an alcohol dose that sustains significant blood levels of ethanol. J. Nutr. 119: 2038-2040. Lieber, C. S., DeCarli, L. M. & Sorrell, M. F. (1989) Experimental methods of ethanol administration. Hepatology 10: 501-510. Lieber, C. S., DeCarli, L. M., Mak, K. M., Kim, C. I. & Leo, M. A. (1990) Attenuation of alcohol-induced hepatic fibrosis by polyunsaturated lecithin. Hepatology 12: 1390-1398. Lowry, O. H., Rosebrough, N. J., Farr, A. L. &. Randall, R. J. (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 193: 265-275. Nanji, A. A., Mendenhall, C. L. & French, S. W. (1989) Beef fat prevents alcoholic liver disease in the rat. Alcohol. Clin. Exp. Res. 13: 15-19. Ponnappa, B. C., Hoek, J. B., Sarchet, K. & Rubin, E. (1986) Dietary carbohydrate level determines the effect of long-term ethanol ingestion on rat pancreatic amylase content. J. Lab. Clin. Med. 107: 556-562. Porta, E. A., Koch, O. R. & Hartroft, W. S. (1972) Recovery from chronic hepatic lesions in rats fed alcohol and a solid super diet. Am. J. Clin. Nutr. 25: 881-896. Rao, G. A. & Larkin, E. C. (1984) Alcoholic fatty liver: a nutritional problem of carbohydrate deprivation and concomitant ethanol ingestion. Nutr. Res. 4: 903-912. Rao, G. A., Riley, D. E. & Larkin, E. C. (1986) Role of dietary carbohydrate in the prevention of alcohol-induced fatty liver. Biochem. Arch. 2: 261-265. Rao, G. A., Riley, D. E. & Larkin, E. C. (1987) Dietary carbohydrate stimulates alcohol diet ingestion, promotes growth and prevents fatty liver in rats. Nutr. Res. 7: 81-87. Rao, G. A., Nishimura, C. Y., Larkin, E. C. & Sankaran, H. (1988) Inverse relationship of blood alcohol levels to energy availability in chronic alcoholic rats. Biochem. Arch. 4: 1-6. Rubin, E. & Rottenberg, H. (1982) Ethanol-induced injury and adaptation in biological membranes. Fed. Proc. 41: 2465-2471. Saab, J. E., Godfrey, P. M. & Brannon, P. M. (1986| Adaptive response of rat pancreatic lipase to dietary fat: effects of amount and type of fat. J. Nutr. 116: 892-899. Sankaran, H., Deveney, C. W., Goldfine, I. D. & Williams, J. A. (1979) Preparation of biologically active radioiodinated cholecystokinin for radioreceptor assay and radioimmunoassay. J. Biol. Chem. 254: 9349-9351. Sankaran, H., Nishimura, C. Y., Lin, J. C., Desai, A., Deveney, C. W., Larkin, E. C. & Rao, G. A. (1989a) Reversal by glucose of pancreatic amylase insufficiency in chronic alcoholic rats. Pan creas 4: 107-111. Sankaran, H., Nishimura, C. Y., Lin, J. C., Larkin, E. C. & Rao, G. A. (1989b) Regulation of pancreatic amylase by carbohydrate in chronic alcoholic rats. Pancreas 4: 733-737. Schick, J., Versphol, R., Kern, H. & Scheele, G. (1984) Two distinct adaptive responses in the synthesis of exocrine pancreatic en zymes to inverse changes in protein and carbohydrate in the diet. Am. J. Physiol. 247: G611-G616. Schmidt, D. N. & Pandol, S. J. (1990) Differing effects of ethanol on

Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1884/4769466 by Tulane University Medical Library user on 19 January 2019

was similar in both control and alcohol groups and was not affected by high BAL. The reduction in amylase response to stimulation is not due to high BAL but rather is due to ingestion of low carbohy drate by alcoholic rats. This is confirmed by the ob servation that, despite high BAL, rats ingesting a higher proportion of carbohydrate responded to cholecystokinin-octapeptide stimulation with an in crease in magnitude; the shift in amylase dose-re sponse curve for cholecystokinin-octapeptide ap proached that of rats ingesting control diet. These stimulatory responses indicate that the cholecystokinin-receptor binding and subsequent stimulus-secretion mechanisms are not significantly altered by high BAL. They also suggest that high alcohol intake accompanied by alcoholemia does not affect pancreatic acinar membrane integrity involving cholecystokinin receptors. To conclude, data from this study indicate that neither alcohol ingestión ñorBAL can be implicated for the reduction in pancreatic amylase activity in chronically alcoholic rats. The results further em phasize the regulatory role of dietary carbohydrate on pancreatic amylase activity and release in chronically alcoholic rats, similar to that described in rats fed diets without alcohol.

ET AL.

BLOOD ALCOHOL

AND PANCREATIC

1891

acini isolated from rats given an ethanol diet. Life Sci. 37: 1359-1365. Wicker, C. & Pugserver, A. (1987) Effects of inverse changes in dietary lipid and carbohydrate on the synthesis of some pan creatic secretory proteins. Eur. J. Biochem. 162: 25-30. Yonekura, I., Nakano, M., Nakajima, T. & Sato, A. (1989) Dietary carbohydrate intake as a modifying factor in the development of alcoholic fatty liver. Biochem. Arch. 5: 41-52.

Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1884/4769466 by Tulane University Medical Library user on 19 January 2019

in vitro stimulated pancreatic enzyme secretion in ethanol-fed and control rats. Pancreas 5: 27-32. Snook, J. T. (1971) Dietary regulation of pancreatic enzymes in the rat with emphasis on carbohydrate. Am. J. Physiol. 221: 1383-1387. Tsukamoto, H., Kiefer, M. A., Rao, G. A., Larkin, E. C, Largman, C. & Sankaran, H. (1985) Cholecystokinin-induced secretion and synthesis of amylase and cationic trypsinogen by pancreatic

ENZYMES

Carbohydrate intake determines pancreatic acinar amylase activity and release despite chronic alcoholemia in rats.

Adverse effects observed in alcoholic rats are often attributed to alcohol per se. Alcoholic liver damage, however, can be avoided by modulating nutri...
1MB Sizes 0 Downloads 0 Views