Intestinal mucosa in diabetic rats: Studies of microvillus membrane composition and microviscosity

Intestinal

Mucosa in Diabetic Rats: Studies of Microvillus Composition and Microviscosity Glenn R. Gourley.

Helen

A. Korsmo,

and

Ward

Membrane

A. Olsen

In experimental diabetes, a number of intestinal brush-border hydrolases and transport systems are stimulated. In this study, we assessed possible effects of diabetes on the composition and membrane fluidity of rat intestinal brush-border membranes that might correlate with these functional changes. We found similar proportions of lipid and protein in the diabetic and control preparations, although there was a considerable increase in total membrane from the diabetic rats, presumably reflecting mucosal hyperplasia. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein revealed an increase in the bands corresponding to sucrase-isomaftase. consistent with an increased enryme activity of sucrase. Membrane lipid analysis revealed only a decrease in fatty acids of the neutral lipid fraction of diabetics-a change that may well have occurred during membrane preparation. 1-B-Diphenyl-1.3.5-hexatriene fluorescence polarization data, obtained as a function of temperature, was similar for the diabetic and control rats, with a three-phase linear model superior to one- and two-phase linear or quadratic models. The overall composition of the intestinal brush-border membrane, unlike other plasma membranes, appears little affected by experimental diabetes.

T

HE LUMINAL (microvillus (MVM) or brushborder) plasma membranes of the enterocytes that line the small intestine are highly differentiated to regulate the digestion, absorption, and secretion of essential nutrients and other substances. Experimental diabetes has been shown to stimulate the functional capacity of these membranes, with increases in the enzymatic activity of a variety of brush-border hydrolases’.’ and stimulation of a number of transport systems believed to reside in part in this membrane.5-9 Aside from a recent paper indicating increased terminal galactose groups of high-molecular-weight peptides of brush-border membranes prepared from rats with chronic diabetes,” we know of no studies that looked for possible effects of diabetes on the composition or structure of the intestinal brush borders that might correlate with the functional changes. The effects of diabetes on other plasma membranes have been studied; for example, diabetes has been shown to alter the composition of membrane lipids in hepatic plasma membranes and erythrocyte “ghosts”.” In addition, insulin is known to alter the microviscosity or membrane fluidity of hepatic,” erythrocyte,13 and bacterial cell membranes,14 and conceivably regulates glucose transport in the adipocyte by altering membrane microviscosity.” These effects on membrane fluidity may have important functional consequences, since changes in the lipid microenvironment of membrane-located enzymes can be reflected in the activity of these enzymes.‘63’7 We, therefore, measured the major constituents of brush-border membranes prepared from diabetic rats, in particular to look for evidence of the altered lipid composition reported for other plasma membranes.” Further studies of these membranes utilized fluorescence polarization techniques. MATERIALS

AND

We studied male Sprague-Dawley 160 g each. Diabetes was produced

A&tabdism,

METHODS rats weighing approximately by an intravenous injection of

Vol. 30, NO. 11 (November).

1983

streptozotocin (Upjohn Co, Kalamazoo, Mich), 70 mg/kg in 0.05 mol/L sodium citrate buffer, pH 4.5, after an overnight fast. The rats were housed in metabolic cages and 24-hour urine collections were tested for glucose (Clinistix; Ames Co, Division of Miles Laboratories Inc, Elkhart, Ind). Only those rats with heavy glucosuria (500 mg/dL or more), which indicates a blood glucose at the time of sacrifice of 300-500 mg/dL,’ were used for study. Control animals were similarly fasted for 24 hours. but not injected. After fasting, all rats were given access to standard laboratory food and water. Five days later, the animals were killed after an overnight fast by stunning and decapitation. The small intestine from the ligament of Treitz to the cecum was removed, flushed with ice-cold saline, everted. rinsed with saline and scraped with a glass slide over ice to remove the mucosa. Mucosal scrapings from groups of IS animals were pooled and the brush borders prepared in sodium EDTA by the method of Forstner et al.” The final wash was in 5 mmol/L sodium EDTA, I mmol/L TRIS-Hepes, pH 7.5. rather than in 2.5 mmol/L EDTA. The brush borders were then suspended in 100 mmol/L mannitol, I mmol/L TRIS-Hepes. pH 7.5, and used to prepare microvillus membranes by the method of Hopfer et al” or Kessler et al.‘0

Lipid Analysis For analysis of membrane lipids, the final membrane pellet was washed three times by suspension in 30 mL of water at 4“C. followed by centrifugation at 24,000 x g. The washed pellet was suspended in l-2 mL of cold water to which sodium chloride was added to a final concentration of 1.7 mol/L.” Twenty volumes of chloroform/methanol (]:I, vol/vol) were added and the membranes homogenized in a glass-Teflon homogenizer at 1000 rpm for three minutes over ice. To minimize lipid degradation, membrane prepa-

From the Gastroenterology Research Laboratory, William S. Middleton Memorial Veterans Hospital. and the Departments of Pediatrics and Medicine, University of Wisconsin, Madison, Wis. Received for publication March 15, I983. Supported by grants AM 13927 and 5 F32HDO5873 from the National Institutes of Health and Veterans Administration Research funds. Dr Gourley is recipient of NIADDK Clinical Investigator Award #I KOB AMOlO??-01. Address reprint requests to Glenn R. Gourley, MD, Department of Pediatrics, University of Wisconsin Hospitals, 600 Highland Ave. Madison, WI 53792. 0 I983 by Grune & Stratton, Inc. 002&0495/83/321 J-0007$01.00/0 1053

1054

GOURLEY, KORSMO, AND OLSEN

ration and extraction were carried to this point before any overnight storage. The glass homogenizer tube was sealed with foil and stored overnight at 4 “C. The method of Suzuki was used to extract and partition the lipids to give a washed lower phase containing most neutral lipids and a dialyzed upper phase containing most of the gangliosides.” The upper phase was concentrated to 2 mL under N2 at 35 OC, dialyzed at 4 OC for 24 hours against 4 L of water with 2-3 changes and then stored frozen with N, at -20 “C until thawed for assay for sialic acid*’ and total sugar? using sialic acid and glucose standards, respectively. Ganglioside or GM, (the predominant ganglioside found by us by thin-layer chromatography) was calculated on the basis of one sialic acid residue per molecule and glycosphingolipid with a mean molecular weight of 846 on the basis of one glucose residue per molecule. The lower phase was concentrated in a rotrary evaporator to about 5 mL and applied to a 2 x 7.5-cm silicic acid column (SIL-LC, 325 mesh from Sigma Chemical Co, St Louis, MO) prerinsed with chloroform for separation of lipids2’ Neutral lipids were eluted with 250 mL chloroform, glycolipids with 1000 mL acetone, and phospholipids with 250 mL methanol. The fractions were concentrated to 3 mL with a rotary evaporator and stored at -20 OC under N, until analysis. The neutral lipid fraction was assayed for total lipidz6 and cholesterol” in triplicate. An aliquot containing 500 wcg of neutral lipid was dried under N,, solubilized with chloroform, and applied as a l-cm streak to a silica gel G-25 plate, which was developed with petroleum ether/diethyl ether/formic acid (80:20:2, vol/vol) with iodine vapor used to detect the spots. **The following standards were usedfor the chromatography: cholesterol oleate (Applied Science Labs, State College, Pa), tri-palmitin (Supelco Inc. Bellefonte Pa), oleic acid (Sigma Chemical Co, St Louis, MO) and sn-glyceryl I- 1, 2-dipalmitin (Supelco Inc, Bellefonte, Pa). The glycosphingolipid fraction was assayed for hexose24 .m triplicate. The phospholipid fraction was assayed for phosphoru? to quantitate phospholipids and hexose to measure glycolipids in the fraction.24 Phospholipids were calculated using a mean molecular weight of 788.

Fluorescence

Polarization

The lipid-soluble fluorescent probe l.6-diphenyl-l,3,5-hexatriene (DPH; Aldrich Chemical Co, Inc, Milwaukee, Wis) was used as a 2 mmol/L stock solution in tetrahydrofuran. In a typical experiment, microvillus membranes (containing 50 rg protein per milliliter) in phosphate-buffered saline were incubated with DPH (final concentration 4 x 10m3 mmol/L). Each determination was made using membranes prepared from pooled mucosal scrapings of the proximal half of the small intestine from four rats. The polarization of fluorescence, P, was measured and interpreted as previously described,” with the parameter ((r”/r)I)-’ directly proportional to the rotational relaxation time, providing a quantitative index of the resistance of the environment to rotational motion of the probe;3’ r represents anisotropy, r” the maximal limiting anisotropy, 0.362 for DPH.” The greater the value of r or ((r’/r)I)-‘, the higher is the apparent microviscosity of the environment in which the fluorophore is located. In this way, microviscosity can be quantitated.” All fluorescence polarization measurements were made using freshly prepared membranes. One-hour long incubations of fluorophore and MVM at 37 “C were compared with short incubations of ten to I5 minutes. Since there is some question about the shape of the resulting function when log P is plotted v (temperature)-‘, four models (linear, two-phase linear, three-phase linear, and quadratic) were compared in their ability to fit the data obtained. A similar approach to testing models and their adequacy using a computer program based on the theory developed by Hudson” has been previously

reported.M This program has been filed with the Biomedical Computing Technology Information Center, Vanderbilt Medical Center, Nashville, Tenn. F-tests were performed to decide if a two-phase was superior to a linear model and if a three-phase was superior to a two-phase model. Breakpoints and anisotropy parameters were compared in diabetic and control membranes using both the twoand three-phase models, and ((r’/r)I)-’ was determined at 25 OC and 37 OC for both models. Statistical comparisons utilized one-way analysis of variance (ANOVA).

Other Methods Protein was determined by the method of Lowry et al,“4 sucrase and trehalase by the method of Dahlqvist,3s and phospholipase A as described by Subbaiah and Ganguly.‘6 The total membrane carbohydrate using glucose standards was determined by the sugar technique described by Dubois et al.24 Membrane proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.” Membranes were solubilized in 0.01 mol/L sodium phosphate buffer, pH 7.1, containing 1% SDS by heating to 100 “C for three minutes. After cooling, 2-mercaptoethanol was added to a final concentration of 5%. The samples were then applied to horizontal thin-layer gels of 5% acrylamide and electrophoresed using a 0.1 mol/L phosphate buffer containing 0.1% SDS. Gels were stained with Comassie blue, photographed, and the negatives scanned at 600 nm using a Gilford 240 spectrophotometer equipped with a model 2424 vertical indexing film adapter and a model 2410s linear transport (Gilford Instrument, Oberlin, Ohio). The area of each of the two subunits of sucrase-isomaltase was determined by planimetry. We also studied the carbohydrate constituents of a single membrane glycoprotein-sucrase-isomaltase. The papain-solubilized protein was purified from brush borders by the technique of Kolinska and Kraml’* to homogeneity by electrophoretic and analytic ultracentrifugational criteria as described by us previously.“.” We purified the protein in triplicate from groups of control and diabetic animals (six purifications, each from a group of 30 animals). Carbohydrate moieties were hydrolyzed to monosaccharides by methanolysis of 0.2 mg quantities of sucrase-isomaltase at 65 “C4’ with mannitol and arabinitol added as internal standards. Trimethyl silyl derivatives were prepared and assayed@ using a column packed with 3% SE-30 on Chromosorb W (Supelco Inc, Bellefonte, Pa) with a Packard Model 419 gas chromatograph (Packard Instrument Co, Inc, Downer’s Grove, Ill) equipped with a flame ionization detector. The area of each peak was determined by planimetry and molar adjustment ratios for fucose, xylose, mannose, galactose, glucose, N-acetylglucosamine, and N-acetylgalactosamine determined experimentally. RESULTS

AND DISCUSSION

A valid comparison of brush-border membranes between control and diabetic animals depends upon a similar degree of purification of the membranes. We found a purification factor for the brush-border membrane enzyme sucrase (specific activity in membranes/specific activity in mucosal homogenates) of 31 + 2.4 (SEM) in eight preparations from control animals and 27.3 + 1.7 in eight preparations from diabetics (difference not significant by Student’s t test), suggesting comparable purification. The composition of these membranes is shown in Table 1. We found twice as much membrane protein in diabetics as

DIABETIC MICROVILLUS MEMBRANES

1055

Table 1. Effect of Diabetes on Brush-Border Membrane Composition

COtWOk

Significance of Difference (P)

Diabetics

Total membrane protein (mgl

1 1.8 _t 2.1

(8)

25.3 + 2.7

18)

Ltpid (mg/lOO mg protein)

92.2 * 10.2 (7)

83.6 + 9.0

(7)

NS

31.8

NS

Glycosphingolipids (@mol/ 100 mg protein) Ganglioside (bmol/ 100 mg protein) Neutral liptds (mg/lOO mg protein) Cholesterol (mg/ 100 mg protein) Phospholipids (mg/lOO mg protein) Cholesterol/phospholipid (wt/wt)

i-O.01

+ 5.8

(7)

30.4 k 3.3

(7)

1.7 i 0.3

(8)

1.2 * 0.1

17)

NS

t 3.0

(8)

30.1 + 2.7

(71

CO.01

15.5 f 0.8

(8)

14.7 k 1.1

(7)

NS

21.5

(8)

28.1

(6)

NS

.04 (6)

NS

44.2

+ 4.6

.98 +

.21 (8)

Carbohydrate (mg/ 100 mg protein)

13.1 + 0.9

Sucrase actwlty (pmoles sucrose hydrolyzed per minute per milligram

i 4.2

.56 2

13.3 2 0.9

(7)

NS

1.65 + 0.14 (8)

3.12

(8)

-Co.00 1

0.96

0.96 f 0.02 (51

(9)

r 0.05

protein) Trehalase actwty (fimoles trehalose hydrolyzed per minute per milli-

f 0.11 (5)

NS

gram protein) Values are given as the mean + SEM. The number of preparations, each from 15 animals, is given in parentheses. Significance of difference was determined by Student’s 1 test. P values greater than 0.5 are indicated as NS (not significant).

controls, suggesting either an increase in membrane protein concentration or an increase in the total amount of membrane. Since the proportion of total lipid and carbohydrate to protein was not affected by diabetes, it is apparent that the finding represented an increase in the total amount of membrane. Despite the remarkable changes reported in the function of the brush-border membrane in diabetes,‘-9 we found little alteration in the composition of the membrane. Table 1 demonstrates the results of our lipid analyses. Our measurements of individual lipid classes are generally consistent with other studies of brush border membranes in rats and other animals.4348 This membrane is known to be especially rich in cholesterol and glycosphingolipids. It is clear that diabetes did not greatly alter the level of individual lipid classes. We did, however, find a decrease in total neutral lipids. In order to determine the nature of the altered lipids in the neutral lipid fraction, we chromatographed aliquots on silica gel G-25 plates. Figure 1 demonstrates the large amount of fatty acids found in samples from control animals compared with samples from diabetic animals. Thus, the decreased total neutral lipids in the diabetic membranes appears to be the consequences of differences in fatty acids. Similar high levels of fatty acids in brush-border membranes have been observed by others43,46.47and presumably are the result of activation of phospholipase activity. Although our procedures were chosen to try to minimize phospholipid hydrolysis by intestinal phospholipase,” it seems likely that phospholipid hydrolysis accounted for much of the fatty acids. Since it is conceivable that differences in phospholipid hydrolysis between control and diabetic membranes reflected differences in phospholipase activity, we measured the activity of phospholipase A in mucosal homogenates from control and diabetic animals. Homogenates from control animals hydro-

Fig. 1. Thin-layer chromatograph of neutral lipids from intestinal brush-border membranes of (a) control and (bj diabetic rats. Neutral lipid standards (c) include: 0 = origin, 1 = diglyceride. 2 = cholesterol, 3 = oleic acid, 4 = triglyceride, and 5 = cholesterol oleate; 500 pg neutral lipid applied for controls and diabetics.

GOURLEY, KORSMO, AND OLSEN

Fig. 2. SDS-polyacrylamida gel electrophoresis of rat intestinal brush-border membrane proteins from three contol fc) and three diabetic (d) preparations. Sl and S2 = sucrasa-isomaltasa subunits. Brush-border membrane protein (50 fig) was applied to each sample wall.

lyzed 0.431 pmol of lecithin per hour per milligram of protein + 0.019 (SEM), n = 5, compared with 0.248 + 0.046 pmol, n = 5, in diabetics (P -C0.01 by Student’s t test). We suspect, therefore, that the decreased fatty acid content of the diabetic membranes is artifactual and related to less phospholipid breakdown during membrane preparation. Table 1 also demonstrates that although sucrase activity was increased in diabetic membranes, trehalase activity was not. Thus, the functional stimulation of brush-border hydrolytic proteins is selective. SDSpolyacrylamide gel electrophoresis of brush-border proteins also suggests a certain specificity of the diabetic effect. As shown in Figure 2, the bands corresponding to the subunits of sucrase-isomaltase protein were increased in diabetic membranes (mean area by

densitometry and planimetry was 1.69 t 0.21 sq in in controls versus 2.87 r 0.11 sq in in the diabetics, P -c 0.01, for Sl; the increase in area for S2 did not quite reach statistical significance). There appeared to be little or no difference in the other bands, however. Because Jacobs, using a galactose oxidase-aodium borohydride technique, found increased terminal galactose groups in high-molecular-weight brush-border membrane proteins from rats with diabetes,” a finding that might account for the decreased rate of degradation of one of these proteins, sucrase-isomaltase, found in diabetic ratsJ9 we purified this protein to homogeneity in order to determine the monosaccharide constituents. As shown in Table 2, we found no significant alteration in the sugars of this protein. Although this may seem inconsistent with the results reported by Jacobs,” it should be noted that he studied membranes prepared from animals with chronic diabetes (7 weeks), while our animals had been injected with streptozotocin five days before study. Thus, it seems likely that the duration of diabetes is related to the difference in protein glycosylation. The fluorescence polarization (P) data was examined using Arrhenius-type plots of log P as a function of reciprocal temperature. To determine the adequacy of the four models being tested to fit these data, residual plots were made. In order for a model to be adequate, there should be no pattern to the residuals plotted, that is, they should be completely random. The residual plots for the linear model were consistently parabolic in shape. Likewise, the two-phase model was generally M-shaped. The three-phase and quadratic models both showed no pattern to their residual plots. The three-phase linear model demonstrated the lowest residual sum of squares in 19/20 data sets. The quadratic model was second in all cases except one, in which it showed the lowest residual sum of squares. F-tests were performed to further compare models. In all cases, the three-phase model was a significantly better fit than the two-phase model. The quadratic model could not be compared with the segmented

Table 2. Effect of Diabetes on the Monosaccharide Composition of Sucrase-lsomaltase Protein Significance COlltrOlS* Monosaccharide

(pglmg

Diabetics’

proteinl

(pg/mg

protein)

IP)

Fucose

26.9

+

3.8

Xylose

24.1

+

2.2

Mannose

34.3

+

6.1

39.0

Galactose

33.7

+

6.4

32.3

+_ 8.1

NS

Glucose

65.0

r 41.7

19.4 +

N-acetylglucosamine

57.1

‘- 15.8

52.9

N-acetylgalactosamine

16.8 k

1.3

21.9

of Difference

*

6.0

NS

9.1 +

3.8

NS

+_ 9.7

NS

3.4

NS

+ 15.9

NS

15.4 *

5.4

NS

Values are given as the mean f SEM. Significance of difference between the means was assessed by Student’s t test. P values greater than 0.05 are indicated as NS (not significant). ‘Each purification was done in triplicate from groups of 30 controls or diabetics.

DIABETIC MICROVILLUS MEMBRANES

Table 3.

Fluorescence

Polarization

1057

IPJ Data as a Function of Reciprocal

Temperature

in Brush-Border

Membranes

from Control and

Diabetic Rats. ControlRats Model

Dfabetlc Rats

Short

Long

Short

Long

Incubation

incubation

Incubation

Incubation

Number leach pooled from 4 rats)

5

4

6

5

2-Phase Transition Temperature (“C)

22.7

+ 2.1

24.3

+ 1.2

23.7

_+ 1.4

23.8

+ 0.6

3-Phase TransItion Temperature - 1

27.8

+ 4.1

28.3

+ 1.7

28.2

_r 2.1

28.4

f 2.5

3-Phase Transition Temperature - 2

15.7 + 5.7

17.9 i 2.3

17.1 * 2.9

17.6 ? 2.5

Values are given as the mean t SD based on a two- and three-phase “best fit” model of the log P versus (temperature)-’

data. There is no significant

difference in breakpoint temperature between the diabetic and control rats using either model (ANOVA).

Table 4.

((r”/r)

-

l)-‘,

which is Proportional

to Microviscosity,

in Control and Diabetic Rat Brush-Border

Control Rats MO&l

Membranes

Diabetic Rats

Temperature

Short

Long

Short

Long

(“C)

Incubation

Incubation

Incubation

lncubatlon

Number (each pooled from 4 rats)

5 2.76

4

i 0.16

2.76

6

25 37

1.71 * 0.03

1.72 + 0.09

1.70 + 0.09

1.71 + 0.12

3-Phase

25

2.74

2.70

2.71

2.68

37

1.69 + 0.04

k 0.15

? 0.08

2.76

5

2-Phase

? 0.07

1.71 + 0.09

+ 0.13

2.73

? 0.11

1.70 + 0.08

+ 0.21 + 0.17

1.71 * 0.12

Values are given as the mean % SD based on a two- or three-phase “best fit” model of the log fluorescence polarization versus (temperature) -’ data. There is no significant difference

between the diabetic and control rats using either model IANOVA).

models using the F-test. Since previous data exist regarding a two-phase model3’.49 and since a threephase model appears superior, both of these models were used to analyze data obtained from fluorescence polarization studies on intestinal brush-border membranes from control and diabetic rats. Highlights based on these data are presented in Tables 3 and 4. Table 3 demonstrates transition temperatures, ie, temperatures at which the segmented log P versus (temperature)-’ function abruptly changes slope. The transition temperatures show no significant differences between control and diabetic brush-border membranes, whether the two- or three-phase models are used (ANOVA). These temperatures are in agreement with those observed in normal Sherman rats.49 In Table 4 we have calculated the anisotropy parameter ((r’/r) - 1) ~-‘, which is directly proportional to the

ability of the brush-border membrane to resist rotation of the fluorescent probe and, hence, is a way of quantitating microviscosity. At both arbitrarily chosen temperatures, there is no significant difference between the controls and the diabetics (ANOVA). These data indicate that diabetes does not alter the enterocyte brush-border membrane sufficiently to affect DPH fluorescence polarization determinations. Thus, diabetic brush-border membranes, although functionally much different than those of controls, show little change in either composition or fluidity. ACKNOWLEDGMENT We thank Drs Milton B. Yatvin and John Porter for the use of the microviscosimeter and the gas chromatograph, respectively, Mr John Vorpahl for assistance in operating the microviscosimeter, Dr Kuni Takayama for frequent discussions and advice, and James Freimuth and Jo Schlies for technical assistance.

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