A~MEIZICAN ,&JRNAL OF PHYSIOLCXY

Vol. 221, No. 3, September 1971. Printed in U.S.A.

E-ffects of diet on rat intestinal soluble hexokinase

and fructokinase activities

MILTOX M. WEISER, HELEN OUILL, AXD KURT J. ISSELBACHER De$mrtment of Medicine, Harvard Medic~dSchool, Boston OZllS, and Gastrointestinal Lhit, Mussachusetts

General Hospital, Boston, hlassachusetts 02114

WEISER,MILTON M., HELEN QUILL, AND KURT J. ISSELBACHER. effects c~f diet on rat intestinal soluble hexokinase and fructokinase activities. Am. J. Physiol. 221(3) : 844-849. 1971 .-The effects of diet on

rat intestinal hexokinase activity, hexokinase isozymes, and fructo- kinase activity were evaluated by separation of these enzymes with DEAE- cellulose chromatography. Casein noncarbohydrate diets

decreased intestinal hexokinase activity when compared to starved animals. High-fructose diets did not increase hexokinase activity. High-glucose or high-sucrose diets increased hexokinase activity

but only as compared to the casein-fed animals. A major effect of high-glucose diets was a relative increase in jejunal type II hexo- kinase over type I. The increased ratio of type 11 over trpe I hexo- kinase appeared to be confined to the upper absorbing cells of the

villus, suggesting a noninductive effect of high-glucose concentra- tions. 7j6, IV (glucokinase) was never detected. Hexokinase activity w’as consistently higher in the ileum while fructokinase ac-

tivity was higher in the jejunum. Starved animals and casein non- carbohydrate-fed animals had equally low fructokinase activity. All three high-carbohydrate diets (glucose, fructose, and sucrose) increased fructokinase activity, though the increase was higher on

the high-fructose and high-sucrose diets.

hexokinase isozymes; glucokinase; DEAE-chromatography

THERE ARE CONFLICTING REPORTS in the literature on the response of intestinal mucosal glycolytic enzymes to diet. Stifel et al. (16) reported that intestinal hexokinase, glu- cokinase, and fructokinase specific activities were increased in animals fed any diet and showed highest activities in an- imals fed carbohydrate-rich diets. They terrned this an

“adaptive” response and recently Rosensweig, Herman, and Stifel (13) suggested a new human clinical entity in which diarrhea was believed secondary to failure of the adaptive response to increase intestinal hexokinase and glu- cokinase activities. Srivastava et al. (15) initially also sug- gested that fed animals showed a higher specific activity for intestinal hexokinase. However, in later work they proposed that the response to diet was due to an intracellular shift from particulate to soluble hexokinase secondary to a high- glucose concentration in the epithelial cell rather than any fundamental change in enzyme synthesis (10). These work- ers, in contrast to Stifel et al. (16), could not detect any glucokinase, Le., the high-k’,, hexokinase or type IV isozyme of Katzen and Schimke (7).

Except for the studies by Srivastava et al. (15), the re- sponses of these intestinal glycolytic enzymes to diet were

evaluated without separation of the hexokinase isozymes and without separation of specific fructokinase from non- specific hexokinases. This latter separation is important since both fructokinase and hexokinase phosphorylate fruc- tose, although the respective products are different. Fur- thermore, the usual fructokinase assays do not distinguish between fructokinase and hexokinase activity (18, 19). Separation of the hexokinase isozymes is important since they have some tissue specificity and respond differently to diet. In the liver, glucokinase is affected by diet while in other tissues, hexokinase isozyxne type 11 appears to respond to dietary changes (3, 6-8, 15).

Recently, we separated specific fructokinase from in- testinal hexokinase isozymes types 1 and 11 by DEAE- chromatography (19). No significant glucokinase (tyke IV) activity was detected in either rat or guinea pig mucosa. It was also shown that hexokinase contributes significantly to the total fructose phosphorylating activity of the small in- testine, a fact which makes separation of fructokinase from hexokinase essential in evaluating dietary effects. To over- come this latter problem, we developed a modified assay for fructokinase, in which the hexokinase activity is elimi- nated, and which is therefore applicable to crude tissue preparations (18).

The development of these techniques provided an op- portunity to define more accurately the effects of diet on these enzymes. It was found that there is indeed a response of intestinal hexokinase and fructokinase to diet and that these responses are specific for a given dietary sugar. How- ever, the data indicate that: there is no adaptive response for intestinal hexokinase,

MATERIALS AND METHODS

Enzyme assays Hexokinase was determined spectropho- tometrically by the method of Sharma et al. (14). A radio- active assay for hexokinase and fructokinase was also used as previously described (19), primarily for estimating col- umn fractions. However, in crude tissue preparations a new assay for fructokinase was used (18) in which the pH of the enzyme preparation is first brought to 6.0, readjusted after centrifugation to pH 7.4, and the activity then assayed in the presence of 50 mM N-acetylglucosamine. These pro- cedures effectively excluded hexokinase activity leaving fructokinase activity intact (18). Sucrase and maltase were determined according to the methods of Messer and Dahl-

844

by 10.220.33.1 on June 23, 2017http://ajplegacy.physiology.org/

Dow

nloaded from

DIET EFFECT ON INTESTINAL HEXOKINASE, FRUCTOKINASE 845

TABLE 1. @%ct oj diet on rat intestine hexokinase q9ec~~c activity -.--

Starved 3 days

Casei n (non- CHO)

High glucose

High fructose

Entire Small Intestine

12.6 •t 1.38

(10) 9.4 + 0.82

(5 > 13.5 zt 1.43

(5 > ND

Jejunum

11.8 + 0.37

(23 > 9.7 3x 0.33*

(33 > 12.3 ,’ 0.75t

(14) 9.3 zt 0.641

(8)

Ileum

18.7 zk 1.11

w 15.3 & 0.80

(16 > 16.2 & 1.08

(13) 16.1 j, 1.80

(5 >

Values are mean hexokinase specific activities in nanomoles per minute per milligram protein followed by SEM. The numbers of animals tested arc in parcrlthcses. ND = not done. The following effects of diet were compared and found to be significantly different for jejunum and entire small intestine: starved vs. casein, P < 0.001; casein vs. high glucose, P < 0.001; starved vs. high fructose, P < 0.005; high glucose vs. high fructose, P < 0.005. Jejunum differs from ileum in all diets, P < 0.001. The following jejunal values were compared and were found not to differ significantly : starved vs. high glucose, and casein vs. high fructose. Ileal values were found not to differ significantly except for starved vs. casein, P < 0.25. *Three to four days on diet, 9.5 * 0.41; 6-7 days on diet, 97 + 057. t Three to four days on diet, 11.6 & 0.57; 6-7 days on diet 12.4 & 1.60. 1 Three to four days on diet, 10.0 & 2.03; G-7 days on diet 8.6 =t 0.40.

qvist (11) and Dahlqvist (4), respectively. DEAE-cellulose chromatography was performed as previously described (19) Protein was determined by the method of Lowry et al.

(9) . Diets. Rat diets were obtained from Nutritional Biochemi-

cals Corp. The high-protein, noncarbohydrate diet con- tained 86 % casein, 10 % vegetable oil, 4 % salt mixture USP XIV, and complete vitamin fortification. The high- glucose or sucrose diets contained 68 % sugar and 17 % casein, as well as the salt mixture and vitamins described above. In addition, a high-fructose diet of 68 % fructose and 17 % casein plus salt and vitamins was prepared in our laboratory.

Animals were kept in separate cages as well as in groups. Thev were paired and weighed daily. Water was permitted ad l&turn. No significant differences were found among the animals in terms of weight gain or ability to thrive. Some animals were kept as long as 6 weeks on a noncarbo- hydrate diet without apparent ill effects.

Tissue preparation. The usual enzyme preparation was made from male and female rats weighing 175-200 g ob- tained from Charles River Laboratories. The animals were sacrificed by cervical dislocation and the small in- testine was removed. After rinsing the lumen with 150-200 ml of ice-cold saline, the intestine was everted, blotted on paper towels, and the mucosa removed by gentle scraping. The mucosal scrapings were suspended in 2.5 or 10 volumes of 0.01 M potassium phosphate buffer, pH 7.4, containing 5 mM EDTA and 0.5 mM dithiothreitol. After homogeniza- tion with a Potter-Elvehjem motor-driven homogenizer, the material was centrifuged at 105,000 X g for 60 min. Protein concentrations of the enzyme preparations (10: 1, v: w) varied from 3 to 5 mg/ml.

RESULTS

In evaluating the effects of diet on hexokinase and fructo- kinase activity, animals were treated as described by Stifel

et al. (16). All animals were fasted for 3 days and either sacrificed or placed on special diets for 3 days and then sacrificed. Some animals were kept on special diets over 3 days, but they did not appear to show any significant dif- ferences from those sacrificed at 3 days (see footnotes, Table 1). Only soluble hexokinase activity was evaluated, although 50 % of the crude homogenate hexokinase activity is particu- late (19) The function of the particulate hexokinase is not clear and has belen considered not to participate in glycolysis (10). In evaluating hexokinase, the spectrophotometric assay of Sharma et al, (14) was used. Figure 1 summarizes the assay and the linearity of the reaction Operationally, the difference in rate between the cuvette containing 100 rnM glucose and that. with 0.5 1x1~ glucose has been con- sidered an estimate of glucokinase (i.e., type 1Y hexokinase) in those tissues known to contain significant glucokinase activity. In mammalian tissues significant amounts of this activity have been detected only in liver. Small increases in activity with 100 mM glucose can be attributed to type II hexokinase (6). As previously reported (19), there is no significant glucokinase activity in the intestine, nor were we able to detect the emergence of glucokinase activity in those animals on special carbohydrate diets either by assay of crude preparations or by isolation on DEAE-cellulose column chromatography.

Dietary efects on intestinal hexokinase. The data in Table 1 indicate the effects of starvation and three special diets on the levels of soluble hexokinase activity in rat intestine. Animals fed a high-casein, noncarbohydrate diet showed a significant decrease in hexokinase activity compared to fast- ing. No statistically significant increase in intestinal hexo- kinase activity was found in animals fed a high-glucose diet when compared to starved rats. However, there was a highly significant increase in intestinal hexokinase activity of ani- mals on a high-glucose diet when compared to those on the casein noncarbohydrate diet. This hexokinase response to dietary glucose was primarily in the jejunum and was ap- parently quite specific in that a high-fructose diet did not produce an increased hexokinase activity. The hexokinase activity in the ileum ‘was significantly higher than in the jejunum in all animals, irrespective of diet, and was highest

MI/VU ES

FIG. 1. IIexokinase assay. Effect of 2 concentrations of glucose used to differentiate hexokinase from glucokinase activity on rat jejunal crude enzyme preparation.

by 10.220.33.1 on June 23, 2017http://ajplegacy.physiology.org/

Dow

nloaded from

846 WEISER, QUILL, AND ISSELBACHER

r

- Jejunum- -Ileum -

FIG. 2. Hexokinase and fructokinase activity distributions along rat small intestine. Represented here are normal laboratory chow-fed animals (essentially high-sucrose diets),

in the ileum of starved rats. The ileum did not appear to respond to high-carbohydrate diets, but in rats fed a high- casein diet there ‘was a significant decrease in hexokinase ac- tivity compared to starved animals. The unusual distribu- tion of hexokinase is illustrated in Fig. 2, which shows that both specific hexokinase activity and total hexokinase activity steadily increased toward the distal small intestine.

In another series of animals the effects of a high-sucrose diet on hexokinases and disaccharidases were compared (Table 2). The increased response of sucrase and maltase to diet reported by other laboratories (2, 5) was confirmed. However, a casein diet appeared to depress sucrase activity when compared with starved animals. A high-sucrose diet increased the hexokinase activity to the same level as oc- curred with the high-glucose diet and was statistically sig- nificant when compared with the casein, noncarbohydrate- fed animals. Thus, the response of intestinal hexokinase to diet appears to be specific for glucose, or glucose-containing disaccharides.

At least four isozymes of hexokinase are known to exist, and the distribution of these isozymes is partly tissue specific (7). Diet or exposure to substrate appears to effect the type I. isozyme except in the liver where only Qpe 0’ responds to diet (6, 14). In order to evaluate the effect of diet on the molecular species of intestinal hexokinase, we first demon- strated the presence in significant amounts of only two in- testinal hexokinases, types 1 and 11(19). The effect of diet on hexokinase isozymes was evaluated using these isolation techniques. The 105,000 X g supernatant of rat intestinal epithelial homogenates was placed on DEAE-cellulose columns and the activity eluted as previously described (I 9). Figure 3 shows the typical elution pattern. DEAE-

cellulose chromatography not only separated the hexo- kinase types 1 and II, but also separated specific fructokinase from the fructose phosphorylating activity of the hexo- kinases.

The effects of diet on hexokinase isozymes are shown in Table 3. The results are expressed in terms of the percent contribution of type 1 and type 1. to the total eluted hexo- kinase activity. Only in the animals fed a high-glucose diet was there a significant and reproducible change with a decrease in type 1 relative to an increase in type 11, a change reflected in the decreased ratio from 0.45 to 0.64 in the nonglucose-fed animals to 0.28 in the glucose-fed animals.

It remained to be determined whether this change in the ratio of hexokinase isozymes on a high-glucose diet in- volved all the cells of the villus including the crypts or only a select group of cells most directly involved with the ab- sorption of glucose. This possibility was evaluated by com- paring the DEAE-cellulose chromatographic patterns of hexokinase isozymes from superficial and deep mucosal scrapings (Fig. 4). It appeared that the main shift to an in- creased dominance of type 11 occurred in the superficial scrapings. This suggests that the increase in hexokinase ac- tivity on a high-glucose diet was primarily an increase in type 11 hexokinase and was confined to those cells in the jejunum most exposed to the high-luminal glucose concen- trations.

Dietary efects on intestinal fructakinase. In contrast to in- testinal * hexokinase, fructokinase appears to be localized predominantly in the proximal portions of the rat intestine (Fig. 2). The response of jejunal fructokinase to diet was evaluated in crude enzyme preparations with a new assay

TABLE 2. E$Ect of diet on rat jq’una/ enzymes

Diet Hexokinase Sucrase Maltase

--

Starved 3 days 11.8 + 0.37 0.13 zt .009 0.28 zt 0.031

(23 1 (3 > (3 > Casein (non- 9.7 zt 0.33 0.08 zt ,004 0.29 41 0.013

CHO) (39) (3) (3) High sucrose 14.0 #z 0.66 0,18 + ,017 0.50 zt 0.033

(28 > (3) (3 >

Values are mean specific activities in nanomoles per minute per milligram protein followed by the SEM. The numbers of animals tested are in parentheses. Statistics for hexokinase : starved vs. high-sucrose diet, P < ,025; casein vs. high sucrose, P < .OOl.

IO 20 30 40

CUL UMN FRAC T/ON

FIG. 3. DEAE-chromatography elution pattern for rat intestine. Fructokinaye emerges first and has no activity with glucose. Next 2 peaks represent hexokinase isozymes I and II (19). They are active with both fructose and glucose.

by 10.220.33.1 on June 23, 2017http://ajplegacy.physiology.org/

Dow

nloaded from

DIET EFFECT ON INTESTINAL HEXOKINASE, FRUCTOKINASE

which eliminates hexokinase contributions to fructose phos- TABLE 4. Effect of diet on rut jejuna~ fructokinase --

847

phorylation (18). The data obtained are given in Table 4 and indicate a fairly specific response to fructose. In con- trast to intestinal hexokinase, fructokinase was not de- pressed by a high-casein noncarbohydrate diet. A high- glucose diet appeared to increase fructokinase activity but not to the extent seen with a high-fructose or high-sucrose diet. That this response of fructokinase to a high-fructose diet was specific for fructokinase was also demonstrated by data obtained with the DEAE-column chromatography (Table 5) By determining the percent contribution of iso- lated fructokinase and hexokinase to the total fructose

Diet

Starved 3 days

Casein (non-CHO)

High glucose

High fructose

High sucrose

Fructokinase

PhosPhorvlatinrr activitv eluted from the column (see Fip.

9.4 * 0.55

(6 > 10.0 rt 0.48

(25 > 12.6 + 0.58

(6) 16.3 zt 1.97

(8 > 16.7 =t 1.40

I (11)

i), ii was’ found that hkxokinase accounted for 60170 % of Values are mean fructokinase specific activities in nanomoles per

the total activity when the animal was starved, or was fed minute per milligram protein followed by the SEM. The numbers of

a high-casein or high-glucose diet (Table 5). Only a high- animals are in parentheses. The following effects of diet were found to differ significantly : starved or casein vs. high glucose, P < .025 ;

fructose diet reversed this to the point where fructokinase starved or casein vs. high fructose or high sucrose, P < ,001. The

accounted for 66 % of the total fructose phosphorylating following effects were found not to differ significantly: starved vs.

activity. This reversal may be explained by the data show- casein, or any of the high-carbohydrate diets compared to each other.

ing that a high-fructose diet increased jejunal fructokinase activity without increasing hexokinase activity (Table 1).

DISCUSSION

TABLE 5. E$ect of diet on total fructose phosphuryhting activity e&ad from DEAE-column chromatography: percent contribution of isolated specijc fructokinase and nonspeczj% hexokinase

One of the primary problems in explaining any dietary effect on mammalian tissue hexokinase activity is the fact

Diet

Total Fructose Phosphorylating Activity

I Ratio

TABLE 3, E$ect of diet on rat jejunal hexukinase subtypes as From From Fruc tokinase Hexokinase

isolated by DEAE-chromatography Starved 3 days 29 71 0.41

Diet Type I/Type II

Starved 3 days 39/61 Casein (non-CHO) 34/66 High fructose 31/69 High glucose 22/78

Ratio

0.64 0.52 0*45 0.28

Casein (non-CHO) High glucose High fructose

that most cells are continuously in contact with a fluid

The values type I/type II are in percent of total activity eluted having a relatively constant glucose concentration and also that most cells are capable of gluconeogenesis, especially from the column. the liver cell. Sharma et al. (14) explain the hepatic dietary response by indicating that only glucokinase appeared to be affected by dietary glucose. Evidence suggests that this effect may be controlled by insulin (14). Brown et al. (3) confirmed the response of liver glucokinase to the nutritional state in both dog and man. They used the starch-gel elec- trophoretic separation of hexokinases developed by Kat- zen et al. (7) and were able to show that this glucokinase was the type IV hexokinase isozyme (equivalent to the high- Km hexokinase). Sharma et al. (14) did not find any effect of diet on liver low-Km hexokinase activity. In other tissues,

FASTED HIGH GLUCOSE

I 15 -=- If 85

I

I

-I however, type 11 hexokinase was shown to respond to vari- DEEP ous physiological factors, such as substrate concentration, SCRAP/MS

It starvation, diabetes, and diet (6, 8). It was thus of considerable interest when Stifel et al. (16)

-22 I I

ti ̂

reported that intestinal hexokinase responded to a number II 70 of different diets, including a noncarbohydrate diet, with

variable increases in enzyme activity over that found in starved animals. Furthermore, they described the presence of an intestinal glucokinase which similarly responded to these diets. However, differentiation between hexokinase

COL UMV FRACT/o/vS activity and glucokinase activity was based solely on the

FIG. 4. Patterns of rat intestinal hexokinase isozymes eluted from a reactions obtained with two different concentrations of DIME-column as effected by diet and differentiated as to villus level glucose using a crude tissue extract. No other investigators

by 10.220.33.1 on June 23, 2017http://ajplegacy.physiology.org/

Dow

nloaded from

848 WEISER, QUILL, AND ISSELBACHER

have reported the presence of glucokinase in rat small in- testine based either on starch gel electrophoresis or DEAE- chromatography (7, 8, 15). A recent report from this laboratory also indicated that there was no significant glu- cokinase in rat small intestine (19). In that study careful attempts were made to detect the presence of glucokinase, a relatively stable isozyme. Extremely small amounts of an activity which had kinetic properties suggestive of gluco- kinase were detected, but there was no evidence that this responded to diet either by assay on crude preparation or by DEAE-chromatography.

However, in the present studies a response of rat intestinal hexokinase to diet was partially confirmed. It was different from that reported by Stifel et al. (16) in that it appeared to be more specific with regard to the dietary sugar and the response was of much smaller magnitude. Stifel et al. ( 16) showed a fourfold increase in rat jejunal hexokinase over fasting controls in animals given a high-glucose diet, and all diets were reported to produce increased hexo- kinase activity (although the high-glucose diet showed the largest increase).

As shown in Tables 1 and 2, the response of hexokinase appeared to be specific for glucose and

jejunal . sucrose

diets. The maximum increase was approximately 25-40 % compared to a noncarbohydrate diet, and this increase was not significant compared to the fasting state. Since normal laboratory rat chow contains high concentrations of sucrose, our findings with intestine are comparable to those re- ported with rat liver (14) and adipose tissue (6). Those re- ports showed little change in low-K, hexokinase activity between fasting and normally fed animals. Our findings are also compatible with the data of Potter et al. (12) who showed that in rats adapted to 36-hr fasting periods, liver hexokinase differed from other carbohydrate-metabolizing enzymes in its cyclic variation; at the end of a 36-hr fast, hexokinase activity was at the level of normally fed ani- mals.

In order to define further the effect of dietary glucose on intestinal hexokinase, changes in individual intestinal hexokinase isozymes were investigated (Fig. 3 and Table 3). A relative increase of .type 11 hexoki nase over ty;r(le I was found. These results confirm those of Srivastava et al. (15). However, this change was most apparent in the cells oc- cupying the tip of the villus (Fig. 4). This finding appears to support the contention of Mayer et al. (10) that the ef- feet of the high-glucose d iet is due to the local concentra- tion of the sugar which may alter the reactivity of the en- zyme or its cellular compartmentalization. The changes observed with glucose infusion by Mayer et al. (10) were quite rapid and indicated that they were not due to a true adaptive response , i.e., synthesis of new enzyme. An explanation for our findings could be that the increase in

REFERENCES

1. ADELMAN, R. C., P, D. SPOLTER, AND S. WEINHOUSE. Dietary and hormonal regulation of enzymes of fructose metabolism in rat liver. J. Bid. Cheti. 241 : 5467-5472, 1966.

2. BLAIR, D. G. R., W- YAKIMETS, AND J. TUBA. Rat intestinal su- erase. II. The effects of rat age and sex and of diet on sucrase ac- tivity. Can. J. Biochem. Physiol. 4 I : 917-928, 1963.

3. BROWN, J.,D.M. MILLER, M. T. HOLLOWAY, AND G. D. LEVE.

hexokinase type II wa .s rela demands. However, no

ted to increased glucose-transport evidence exists that glucose is

phosphoryla ted i n transport across th possibilities are that the increased

e intestine glucose co

- (17) * ncen

Other tration

hexokinase in- inhibits a. repressor, alters an endogenous hibitor, or simply stabilizes the enzyme.

An additional feature of intestinal hexokinase is its distri- bution along the small intestine. As shown in Fig. 2 and in Table 1, the distal small intestine always had higher hexo- kinase activity, both total and specific activity. This was in marked contrast to fructokinase activities, which were highest in the proximal small intestine. There is no apparent explana the gut.

tion for this difference The distribution of fr

in enzyme uctokinase

d is

- * istribution along compatible with

a specific induction mechanism for the utilization of dietary fructose by jejunal epithelium of rats on high-fructose or high-sucrose diets.

Da tivity

ta in Table 4 increased sign

show ifican

that i tly on

ntestinal high-fruc

fruc tokinase tose or high

ac- .-su-

crose diets (Table 4). Similar results were obtained by Stifel et al. (16), although their increases were of greater magnitude. Their data also indicated a greater stimulation of activity by high-fructose as compared to high-sucrose diets. We found no significant difference between a high- sucrose or high-fructose diet. We also demonstrated by DEAE-cellulose chromatography (Table 5) that the in- crease in fructoki nase activity a isolated enzvme. The data on

.ppears to be specific intestinal fructokin

fruc that

for the ase are

tokinase by noncarboh

Adel- ydrate

similar to that obtained with liver man et al. (1). They, too, found diets maintained a low liver fructokinase level similar to

tose diet markedly increased this fasting while a high-fruc activity.

In conclusion, the data presented here do not support an adaptive response of intestinal hexokinase to diet, if one infers from that term control of enzyme synthesis. A signifi- cant change in intestinal hexokinase activity in response to dietary glucose was apparent only when compared to a noncarbohydrate diet. The magnitude of this change was relatively small and appeared to be secondary to a local cellular increase in glucose concentration in the actively absorbing cell. In addition, there was no evidence for the presence of an intestinal glucokinase response to diet. Thus, it is clear that more extensive studies are needed on the molecular mechanism responsible for changes in intestinal glycolytic enzyme levels before one can attribute any etiologic role to a maladaptive response of these enzymes in diseased states

This study was supported by National Institutes of Health Grants AM-01392 and AM-03014.

Received for publication 19 March 1971.

Hexokinase isoenzymes in liver and adipose tissue of mwldn and dog. Science 155 : 205, 1967.

4. DAHLJ~VIST, A. Method for assay of intestinal disaccharidases. Anal. B&hem. 7 : 18-25, 1964.

5. DEREN, J.J., S. A. BROITMAN, AND N. ZAMCHECK. Efkt of diet upon intestinal disaccharidases and disaccharide absorption. J. Glin. Invest. 46 : 186-195, 1965.

by 10.220.33.1 on June 23, 2017http://ajplegacy.physiology.org/

Dow

nloaded from

DIET EFFECT QN INTESTINAL I-IEXOKINASE, FKUCTOKTNASE 849

6.

7.

8.

9.

10.

11.

12.

JAMSEM, R., S. J. PILKIS, AND M. E. KRAHL. Properties of adaptive 13. hexokinase isozymes of the rat. Endocrinology 81 : 1397-1404, 1967. KATZEN, H. M., AND R. T. SCHIMKE. Multiple forms of hexokinase in the rat: tissue distribution, age dependency, and properties. 14, Proc. N&l. Acad. Sci. U.S. 54: 1218-1225, 1965. KATZEN, H. M., D, D. SODERMAN, AND C. E. WILEY. Multiple forms of hexokinase: activities associated with subcellular particu- late and soluble fractions of normal and streptozotocin-diabetic

15

rat tissues. J. Biol, Chem. 245: 4081-4096, 1970. LOWRY, 0. H., N. J, ROSEBROUGH, A. L. FARR, AND R. J* RAN- DALL. Protein measurement with the Folin phenol reagent. J. 16 Bid. Chem. 193 : 265-275, 195 1. MAYER, R. J., P. SHAKESPEARE, AND G. H~BSCHER. Glucose metabolism in the mucosa of the small intestine. Changes of 17. hexokinase activity during perfusion of the proximal half of rat small intestine. Biochem. J. 116 : 43-48, 1970. MESSER, M., AND A. DAHLQVIST. A one-step ultramicro method for the assay of intestinal disaccharidases, Anal. 13iochem. 14: 3X--

l8 ’

392, 1966, POTTER, V. R,, R. A. GEBERT, AND H. C. PITOT. Enzyme levels 19. in rats adapted to 36-hour fasting. Aduan. Enzyme Reguhtion 4: 247-265, 1966.

ROSENSWEIG, N. S., R. H. HERMAN, AND F. B. STIFEL. Familial glucose intolerance: a failure of jejunal glycolytic enzyme adapta- tion to dietary glucose. Gastroenttrology 58 : 990, 1970. SHARMA, C., R. MANJESHWAR, AND S. WEXNHOUSE. Effects of diet

and insulin on glucose-adenosine triphosphate phosphotransfer- ases of rat liver, J. Biol. Chem. 238: 3840-3845, 1963. SRIVASTAVA, L. M., P. SHAKESPEARE, AND G. H~~BSCHER. Glucose

metabolism in the mucosa of the small intestine. Biochem. J. 109: 35-42, 1968. STIFEL, F. B., N. S. ROSENSWEIG, D. ZAKIM, AND R. H. HERMAN. Dietary regulation of glycolytic enzymes. I. Adaptive changes in

rat jejunum. Biochim. Bioflhys. Acta 170: 221-227, 1968, WEISER, M. M., AND K. J. ISSELBACHER. Phosphoenolpyruvate- activated phosphorylation of sugars by intestinal mucosa. Biu-

dim. Biophys. Acta 208: 349-359, 1970.

WEISER, M. M., AND H. QUILL. Estimation of fructokinase in crude tissue preparations. Anal. Biochem. In press.

WEISER, M. M., H. QUILL, AND K. J, ISSELBACHER. Isolation and properties of intestinal hexokinases, fructokinase, and N-acetyl- elucosaminekinase. J. Biol. Chem. 246: 2331-2337, 1971.

by 10.220.33.1 on June 23, 2017http://ajplegacy.physiology.org/

Dow

nloaded from