In vitro response of enzyme-dispersed rat intestinal villus and crypt cells to hormones

In odtro response of enzyme-dispersed rat intestinal villus and crypt cells to hormones

Bunting and Best Departmr~nt of .%l~dicul Research, Charles H. Best Institute, Lrniversity of Toronto, Tororzto, Ont., Canada hf5G IL6

Received April 6, 1979

HOFFMAN, A. G . D., and KUKSIS, A. 1979. ltz vitro response of enzyme-dispersed rat intestinal villus and crypt cells to hormones. Can. J. Physiol. Pharmacol. 57, 1393-1400.

Villus and crypt cells isolated from rat jejunal tissue by an improved enzymatic procedure were tested for their ability to respond to hormonal stimulation using several metabolic tracers. Incubation with cyclic AMP, theophylline, isoproterenol. and epinephrine resulted in 20-707; increases in incorporation of D-glucosamine into glycoproteins. The newly formed material was largely (90-955,) recovered from the medium, apparently as a result of active secretion, because tests for cellular leakage and breakage failed to account for it. The above hormones and pharmacological agents as well as glucagon, insulin, and cortisol, however, failed to modify the rate of incorporation of acetate and glucose into the total cellular lipids of the isolated crypt and villus cells. Likewise, segments of intestinal tissue, although readily responding with increased incorporation of D-glucosamine (50-1000;), failed to demonstrate any effect of added hormones on the incorporation of acetate even when lipogenesis was stimulated by addition of glucose or fructose. These findings are in agreement with certain previous studies with intact intestinal tissue in vivo and in vitro, and attest to the suitability of the enzyme-dispersed cells for metabolic studies.

HOFFMAN, A. G . D., et KUKSIS, A. 1979. In vim response of enzyme-dispersed rat intestinal villus and crypt cells to hormones. Can. J. Physiol. Pharmacol. 57, 1393-1400.

On a test6 la capacitd de cellules de villosites et de cryptes provenant de tissus de j6junum de rat et isolkes B l'aide d'une technique enzymatique Cprouvke, B repondre h une stimulation hormonale et ce, en utilisant divers traceurs mktaboliques. L'incubation avec de I'AMP cyclique, de la theophylline, de l'isoproter6nol et de 1'Cpinephrine rCsulte en des augmentations de 20-70(;; de l'incorporation de D-glucosamine dans les glycoprotCines. Le tissu nouvellement form6 est largement recouvert du milieu (90-955: ), ceci resultant apparemment d'une secretion active; en effet, des tests de fuite ou de cassure cellulaire se sont rCvelks n6gatifs. Cependant, les hormones prbcitks ainsi que des agents pharmacologiques tels que le glucagon, l'insuline et le cortisol, sont sans effet sur le taux d'incorporation d'ac6tate et de glucose dans les lipides cellu- laires tcataux des cellules isolees de cryptes et de villosit6s. De m$me, des segments de tissu intestinal qui ont r6pondu positivenlent B une augmentation de l'incorporation de u-glucosamine (50-100(!%,) ne montrent pas d'effet des hormones ajoutkes, sur l'incorporation d1ac6tate et ce, m$me lorsque la lipoginkse est stimulk par l'addition de glucose ou de fructose. Ces r6ultats sont en accord avec certaines Ctudes prkcedentes utilisant des tissus intestinaux intacts in vivo et in vitro, et temoignent du fait yue des cellules dispersies enzymatiquement conviennent B des etudes portant sur le metabolisme.

[Traduit par le journal]

Introduction structural integrity of the plasma membrane also ex-

We have recently developed an improved enzymic tended to the function of the hormone

method for the isolation of rat small intestinal receptors located there. In order to assess further the

mucosal cells from the crypt to villus gradient quality of the cell preparation and its relationship to

(Hoffman and Kuksis 1g79). These cells appeared the intact tissue, it was important to examine its

morphologically normal and exhibited high rates of to known and pharmacological

metabolic activity when compared with intact tissue. agents. Previous work with chelate-dispersed in- It was not certain, however, whether or not the ultra- testinal had to any

response (Shakir et al. 1978). Although it had been ABBREVIATIONS: db CAMP, ~ ~ , ~ " - d i b u t y ~ ~ l adenosine 3', reported that cyclic AMP formation is stimulated by

5'-cyclic rnonophosphoric acid; 8-br CAMP, 8-bromo- several hormones in mechanically dispersed in- adenosine 3',5'-cyclic monophosphoric acid; BSS, balanced testinal cells (De Jonge and Hiilsmann f974), it is salt solution: HEPES, N-2-hydroxyethylpiperazine-N'-2- now known that this response does not need intact ethanesulphonic acid; BSA, bovine serum albumin; BAEE, N-benzoyl-L-arginine ethyl ester. cells and can be obtained with cell homogenates

lAuthor to whom all correspondence should be addressed. (Gaginella et al. 1978). There has been no previous

0008-4212/79/121393-08$01 .OO/O @ 1979 National Research Council of Canada/Conseil national de recherches du Canada

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1394 CAN. J8 PHYSIOL. PHARMACOL. VOL. 57, 1979

work on the hormonal stimuIation of enzyme prepared intestinal cells.

The present study denlonstrates that although intestinal epithelial cells obtained by enzymic digestion do not respond to insulin, glucagon, and cortisol with a, modulation of the lipogenic rate, they do respond to adrenergic hormones and pharmacological agents with increased synthesis of glycoproteins.

Methods and Materials Anirntils

Male Wistar rats weighing 200-250 g were purchased from Canadian Breeding Laboratories, Montreal, and housed under conditions of controlled illumination. They were provided with Purina rat chow and water ad libitum until time of killing.

Materials Glucagon (crystalline, mixture of bovine and porcine),

epinephrine bitartrate, db CAMP (grade 11), 8-br CAMP, theophylline, and hydrocortisone were purchased from the Sigma Chemical Company (St. Louis, MO, U.S.A.). Cholera toxin was obtained from Schwal-tz-Mann (Orangeburg, NY, U.S.A.), isoproterenol from Winthrop Laboratories (Aurora, Ont., Canada), pentagastrin from Ayerst Laboratories (Montreal, P.Q., Canada), and insulin (bovine) from Con- naught Laboratories (Toronto, Ont., Canada).

[I-"CIAcetate (specific activity: 8.76 mCi/mmol; 1 Ci = 37 GBq) was purchased from ICN Canada (Montreal, P.Q., Canada), D-[U-'T]glucose (specific activity: 230 mCi/mmol) from AmershaidSearle Corp. (Don Mills, Ont., Canada), and L-[4,5-"H(N)]leucine (specific activity: 20 Ci/mmol) and D-[I-"C]glucosamine hydrochloride (specific activity: 57 ,uCi/,umol) from New England Nuclear (Boston, MA, U.S.A.) .

All other materials employed during the cell separations and incubations have been described elsewhere (Hoffman and Kirksis 1979).

Cell Prrparution and Incubation Protocol Upper villus, lower villus, and crypt epithelial cells were

isolated from the jejuna of rats, by a combined scraping and enzyme digestion with hyaluronidase and DNase in Hanks' BSS containing soybean trypsin inhibitor (Hoffman and Kuksis 1979). Rats were killed by stunning and decapitation. Cells prepared by this procedure were tested for cross- contamination by comparing the alkaline phosphatase (Weiser 1973) specific activities between villus and crypt cells. We routinely obtained villus to crypt ratios of between 3.9 and 4.5.

For studies with intact tissues, 1-cm intestinal segments were prepared as described by Lukie and Forstner (1972) and 0.8-mm liver slices as detailed by Dietschy and McGarry (1974).

Cells were irlcubated in modified Hanks' BSS containing 15 mlV HEPES buffer, 4% BSA (w/v), and 660 inhib- itory BAEE units/mL of soybean trypsin inhibitor. Tissues were incubated in Kreb's-Ringer's bicarbonate buffer with 4% BSA (Lukie and Forstner 1972). Incubations were as described previously (Hoffman and Kuksis 1979).

Dye Exclusion Test The ability of the enzyme-isolated villus and crypt cells

to exclude 0.2% Nigrosin dye was examined as a function

of time. The dye test was performed as described by Kalten- bach et ul. (1958) with dark-stained nuclei scored as an indicator of permeability to this dye.

Enzyine Analyses Alkaline phosphatase (EC 3.1.3.1 ) activity in cell homog-

enates and in the medium was assayed colorirnetrically at 540 nm by quantitating the hjfdrolysis of p-nitrophenyl orthophosphate substrate, as described by Weiser ( 1973 ) .

Lactate dehydrogenase (EC 1.1.1.27) activity in homogenates and media was determined by recording ultraviolet absorption at 340 nm for the foreward reaction of pyruvate f p-NADH -+ lactate f P-NAD, as previously described (Decker 1977 ) . Hormonal arzd Metabolic Studies

For experiments on glycoprotein synthesis, the medium contained 5 m M glucose, 18 m M D-glucosamine hydrochloride, 8 mM glutamine. and 1 yCi ~-[ l -~~C]glucosamine hydrochloride. For lipid synthesis in isolated cells, the medium contained 10 mM acetate, 5 mM glucose, and 5 pCi[l-l'C]acetate or 1 m M glucose and 5 ,uCi D-[U-'"1- glucose. For studying lipid synthesis in intestinal segments and liver slices, the medium contained 10 mM acetate (5 pCi [1-"Clacetate) and this was supplemented where indicated with 10 mM fructose, 11 m M glucose, or 10 mM pyruvate.

Hormones and pharmacological agents were di5solved in phosphate-buffered saline of appropriate pH (glucagon. pH 9.1; insulin, pH 4.0; d b CAMP, 8-br CAMP, isoproterenol, theophylline, pentagastrin, and cholera toxin, pH 7.4; and epinephrine bitartrate, 0.01 N HCl), and 100 ,uL added to each incubation vessel. Hydrocortisone was dissolved in 70% ethyl alcohol (v/v) with 10 yL added to the apyro- priate flasks. Due to rapid destruction in oxygenated media at 37"C, the addition of epinephrine was repeated every 30 min to ensure continued appropriate titre of this hormone (Logothetopoulos, J. : personal communication). The metabolic effects of the hormones and other agents were meas- ured by the addition of radiotracers in 100 yL of phosphate- buffered saline 30 min after a preincubation of the cells or tissues and hormones. Immediately after incubation, the media were removed and the cells or tissue slices washed once with ice-cold Hanks' BSS.

Glucosan~ ine Incorporation irl to Glycoproteins Cells and intestinal tissue slices were homogenized in 5

mM NapEDTA (pH 7.4) by ultrasonication or by use of a Polytron (Kinematica GmbH, Lucerne, Switzerland). Gly- coproteins were precipitated from homogenates and from the incubation medium by the use of ice-cold 10% trichloro- acetic acid solution containing 1 % phosphotungstic acid (Lukie and Forstner 1972) and 10 mM D-glucosamine hydrochloride. Purified acid-precipitable material was dissolved in 1 N NaOH with aliquots taken for radioactivity determination and protein estimation (Gornall et al. 1949).

Acetate and Glrlcose incorporation into Lipids Cells and tissue slices were disrupted as described above,

in 10 mL chloroform-methanol (2: 1 , v/v) and the lipids from these homogenates and from the media extracted ac- cording to Folch et al. (1957). The lower phases from two extractions were combined, passed through anhydrous sodium sulphate, and the solvent removed at 40°C under nitrogen. The lipids were redissolved in a small volume of toluene-ethanol (2: 1, v/v) for radioactivity determination by liquid scintillation counting in a Searle Isocap 300 instru-

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HOFFMAN AND T<UT<SIS 1395

ment (Searle i%nalytic, Oakville, Ont., Canada). Yn some cases, the total lipid extract was subjected to alkaline n~eth- anolysis with rnethanolis SOCB~LIPI I nasthoxide dC3la.s 1971) and the rezr~lting fatty acid methyl ester? and free sterols p~rrifieal Iiy thin-layer chso~natcdgraphy (Breckenridge and Kuksi5 1967 ,I.

Srcrri.sricta/ Anr1l~sc.s Data for metabolic cxperin~ents wcre calcuIated as narlo-

moles sub5trr1te inccrporated per milligram cell protein per hour, and rsprescnt the mean -t SEM for (hJ) replicate ew- periments. In some cases, the4e were furtIaer corrverted to percentage deviaticdns from control valtres. For comparison o f hormone-treated data versu5 controls, Student's t-test for equal sized paired groups was used (Snedecor and Ca~chran 1967).

Results

The effects of the various adrenergic hormones and related agents on the incorporation of D-glucosamine into cellular and released glycoprotein by isolated rat jejunal upper villus cells are shown in the upper half of Table I . A11 agents, except cholera toxin, caused a significant increase in the incorporation of n-glucosamine into glycoproteins and this was reflected primarily in the material recovered from the medium. On the average, 17.8 76 sf the radioactivity was present in the cells and about 82.2% was released into tlne medium. Tlae overall s t in~~la t ion ranged from 1 19 to B 7096 compared with a control value of 108%. It is shown in the lower half of Table I that all hormones and pharmacological agents, except cholera toxin, stimulated the incor-

Eflect of Adrenergic Horrnoncs on Glycoprotrin poration o f D - ~ ~ u c o s ~ ~ ~ I I ~ into acid-precipitable S,ynfhes.is and Secretion mnnaterial recovered from thc crypt cells and asso- - -

As a. means of standardizing our assay system and ciated medium. as a basis for comparison with isolated intestinal cells, we redetermined thc response of jejunal tissue scganents to horsnonal stimulation. 'The incorpera- tion of D-glucosamimse into acid-precipitable glycoprotein in the presence of various adrenergic hormones and appropriate pharmacological agents is sho~vn in Fig. f . Hsoproterenol and epinephrine caused increases in incorporation of n-glucosarnine of 1 1 2 and 80 % , respectively. When epinephrine and theophylline were combined. the incrcase was 201 % over the control value, presu~nably due to the potentiatinag effect of the purine phosphodicsterase inhibitor. Cholera toxin caused only a naoderate increase (30% ) in glycoprotcin synthesis in vitro. These results are in general agreement with those of Forstner ef ab. ( 1973 ) anad indicate that our assay conditions are adequate.

The overall stimulation ranged from 106% for cycaic AMP to 121 and 127 % for theophylline and isoproterenol, rcspectively, and to 150% for epinephrine compared with 100% for tlne control. Cyclic AMP and cholera toxin had no effect. About 96% of the radioactivity in the glycoprotein precipitate was recovered from the medium and about 4 % from the crypt cells. The overall initial incorporation of label into the crypt cells was about 4.7 ti~zaes that for villus cells, and the difference rose to eightfold for epinephrine-stimulated cells.

As it was possible that the glycoproteins found in the medium were released by cell breakage, we examined the extent of the concomitant release of the cellular enzymes into incubation medium. For this purpose, we followed the appearance of the brush border glycoprotein enzyme, alkaline phos-

r---7 C ~ n t f o i 9 6 4 + + 4 CS

CL-2 d b Theophyli~ne {so - Epinephrine E * T Cholera

CAMP ( T ) pmterenol (E j toxin

FIG. I . ERects of hormones and pharmacological agents on the incorporation of D-[l-~4C]glucosamine into acid- grecipitable glycoproteins from intestinal jejunal segments. Concentrations of each compc~und are identical with those presented in Table I. Control value rcpreserats 0.892 nmol D-glucosarnine incorporated per milligram protein per hour. Data expressed as mean f SEM for N = 3.

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1396 C A N . J. PHYSIOL. PHARMACOL. VOL. 57, 1979

TABLE 1. Effects of hormones and pharmacological agents on the incorporation of D-[l-MC]glucosan~ine into glycoproteins of isolated rat jejunal villus and crypt cellsa

Cell ty Pe Treatment

nmol u-glucosamine incorporated Percentage --- of control

Concn. Cells Medium Combined value i SEM -

Villus Control + db cAMP + theophylline + isoproterenol + epinephrine + cholera toxin

<0.001 <0.001 <0.001 <0.025

N.S.

Crypt Control + db cAMP + theophy lline + isoproterenol + epinephrine + cholera toxin

N.S. <0.$1 <O .005 <0.001

N.S.

aCells preincubated with hormones for 30 rnin and then incubated for 60 rnin with radiotracer. Buffer (5 mL) contained 18 m M D-glucosamine ( I pCi D-[I-14C]-glucosamine), 8 mM glutamine, and 4% BSA.

bData represent mean + SEM for six experitnerltal determinations. Sigrmificanze from control value tested by Student's r-test.

TABLE 2. Changes in cell protein content, membrane permeability, and loss of soluble lactate dehydrogenase and brush border alkaline phosphatase with incubation in Hanks' buffer for

varying lengths of timea

Medium Medium 0.2'x Nigrosin lactate alkaline

ll~cubation time, exposure, dehydrogenase phosphatase Cell type min Cell protein % unstained cells activity, ?{, activity, :/;

Upper villus 0 15 30 60 90

aAll data represent the mean f SD of three replicate experiments. Mediu~n lactate dehydrogenase and alkaline phosphatase activity expressed in terms of cell content at To = 100%.

phatase, into the medium as a function of the incubation time. It is shown in Table 2 that the alkaline phosphatase in the mediuin at 90 min comprised approximately 12 % of the activity initially detected in the villus cells and about 28% of that from the crypt cells. These losses of the brush border enzymes were less than those obtained for a soluble cytoplasmic enzyme, lactate dehydrogenase, from the villus (24% ) and the crypt (43 % ) cells over comparable time intervals. A determination of the cell protein content following incubation showed no significant decrease over 90 min of incubation. How- ever, the ability of the cells to exclude 0.2% Nigrosin dye did show a steady decrease from an initial 90% (villus ) and 80% (crypt) to about 52% (villus and crypt) by the end of the incubation period. Clearly

the limited breakage and (or) leakage of the cells cannot account for the appearance of 82-95% of the labelled protein in the medium of the correspond- ing incubations.

Ef;fect of Hormones on Lipid Biosynthesis As a control for the hormonal regulation of lipid

biosynthesis in the isolated mucosal cells, we determined the hormone respoilse of intact intestinal tissue in vitro, under conditions of lipogenesis stimulated by glucose or fructose. It is shown in Fig. 2 that there werc no significant differences (P > 0.010) between control tissues and tissues exposed to cyclic AMP or glucagon on the rrmetabolic utilization of acetate, either in the absence or presence of glucose in the medium. The addition of glucose to

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HOFFMAN 4 N D KUICSIS 1397

Intestinal segments

Conlrol + + + + + + Control + + Control + Contra + db lnurl~n Gluca- Ep~ne- Penta- Cholera db Gluca- Gluca- Gluca-

CAMP gon phr~ne gnslrtn t ox~n CAMP gon Qon W n

FIG. 2. Incorporation of [I-14CC]acetate into total lipids of intestinal jejunal segments. Segments were incubated in Kreb9s HCO, buffer with 10 m!W acetate + 10 ni!kf fructose (A-G). 10 m M acetate (H-J), 10 m M acetate 4- 11 mM glucose (K, E)? or 10 mM acetate -+ 10 m.M pyruvate (M, N). Concentrations of hormones and pharmacological agents are given in Table 3 with the exception of pentagastrin (10 p M ) . All samples also had 5 pCi of [1-14C]acetate added after a 30-~nin preincubation, with a 60-min main incubation. Llata presented as mean 1: SEM for six replicate determinations.

the medium resulted, however, in a two- to three-fold nificant stimulation or depression of lipid synthesis increase in the overall incorporation of acetate. Like- was observed for either villus or crypt cells. wise, the substitution of pyruvate or fructose as carbon sources had no effect on the horn~onal response. The suitability of the assay system was confirmed in rat liver slices, where cyclic AMP and glucagon caused significant decreases in the overall incorporation of acetate. When glucose was added to the medium the control value was nearly doubled and the inclusion of glucagon or epinephrine resulted in a significant depression of acetate incorporation. Insulin, pent agastrin, and cholera toxin did not alter the rate of acetate utilization.

An absence of metabolic response upon hormone addition to the enzyme-dispersed intestinal epit hclial cells, which is conlparable to that seen with intact tissue in vitro, is demonstrated in Table 3. When lipid synthesis from acetate was assayed at or near substrate saturation using glucagon, insulin, epinephrine, hydrocortisone, cyclic -4MP, or cholera toxin, the responses produced were not significantly different froill control values (P > 0.01 0) . This was true for both villus and crypt cells. When the experiment was repeated with radioactive glucose at less than substrate saturation levels, again no sig-

Discussion

Effect of A drenergic Hormones on Glycaprstein Synthesis

Early work has given largely negative or con- troversial effects with various horn~onal al~odulators of mucosal xnetabolisrn (Levin 1969). However, Forstner et a1. ( 1973 ) demonstrated stixnulation of intestinal glycoprotein synthesis by adrenergic agents in intestinal seginents using D-[l-14C]glucosamine as a tracer. This observation was recently confirmed by LaMont and Ventola ( 19771, who studied glycoprotein synthesis in rabbit colon. In further extension of these findings we were able to modulate the incorporation of D-[ 1 -14C] glucosamine into the glycoproteins of jejunal segments and isolated cells by appropriate pharmacological agents. The degree of stimulation of incorporatioil of D-glucosamine by the isolated cells, however, was significantly lower than that observed in segments of intestinal mucosa. In contrast with the findings of Forstner et al. ( 1973 ) for rat jejunal segments, the bulk of the acid-

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CAN. J. PHYSIOL. PHARMACQL. VOL. 5 3 , 1979

TABLE 3. Effects of hormones and phamn~acological agents oas the iracsrporatic~n of [I-14CJacetate or ~-[$T- 'T]gl~~cose into total lipids of isolated rat jejunal villus and crypt cells"

Incorpcssation of substrate iiato lipids, of control value

Cell type Treatment Concn. [ I -14C]Acetate ~-[U-J~C]d'B~ucose

Villus Control + glucagon + iassuli~~ + epinephrine + cortisol + cyclic AMP + cholera toxin

Crypt Control + glucagota + insulin -+ epinephrine + cortisol + cyclic AMP + cholera toxin

"Cells incubated for I h in 5 mL Ii'lnks' BSS contnirsing either 10 mM ~ c e t a t e (5 &i [I-14C]acetate), 5.5 rn 11 glucose, or 1 r l~ iZ lP glucose ( 5 &i D-[U-'4C]gliicose), plus 4% BSA.

nor^: Data represent mean I SEM for triplicate experiments.

precipitable radioactivity sg~mthesized by the cells was recovered in the medium. As both crypt and villus cell preparations contained 10-2096 goblet cells, it remains uncertain which cells were respon- sible for the increased synthesis of glycoprotein. Previous work has shown that both colonic goblet (Lahkrnt and Ventola 1977) and jejunal epithelial cells from the villus and crypt regions (Weiser % 873 ) incorporate D-glucosamine into gl ycaproteins, which may be secreted from these cells. The marked response of our cells to horrnonal and pharnnacolog- ical agents sl~ows that the appropriate receptor sites survived the method of cell dispersion undarxaaged. These Bindings therefore support earlier reports of adrenergic receptors in intestinal mucosa, based on studies with everted sacs (Aulsebrook % 965 ) .

The lack of significant stimulation of incorporation of D-glucosaminc into villus and crypt cell glycoproteins by cholera toxin was unexpected in view of the well-recognized role that this agent plays in vivs in intestinal adenylate cyclase activation (Kimberg et ~ 2 . 1 97 % ; Schafer st al. 19'70). A similar lack of in vitro effect of this toxin on mucosal cell preparations, however, has been reported by others (De Jonge and Hiilsmann 19'74; Weiser and QuilI 1975), although there was evidence sf binding of I1""labelled cholera toxin to the luminal surface of villus cells (Weiser anad Quill 1975). Apparently, a luminal exposure of the toxin in vivs is necessary be- fore any elevation of cyclic AMP levels can be detected in isolated intestinal epithelial cells (De Jonge 1975; Schwartz et a&. % 9'75).

Efleet of Adrenergic Hsr?~.eonps on GHycopro~ei~z Secretion

As the increases in the glycoprotcin synthesis brought about by the hormones tverc anaa~ifested largely in the material found in the medium, both synthesis and secretion of labelled glycoproteins woa~ld appear to be mediated by the added hormonal agents. The pssibility of grcrss cellular damage was ruled out by monitoring the leakage of appropriate cellular cnzymes and by rneastnrirsg increases in the permeability of the cell merrlhrane to Nigrssin dye. It must, therefore, be concluded that the appearance elf large amounts of radioactive glycoproteins in the rnediurn nskast largely represent an orderly secretion involving one or more of the processes previously demonstrated : the secretion of glucc~samine-containing lipoproteins (Yoaasef and Kuksis 11 972); the release of absorptive cell glycocalyx ( Forstner 1970; Weiser st al. 1978 ) ; and secretion of glyccqx-oteins by goblet cells (1,aMsnt and \Tentola 1977). The present data do not allow us to distingtxish arnong tlmese possibilities. It is suggested that this could be accomplished in the future by a resolution c~f the cell types involved in intestinal glycoprotcina synthesis and secretion. Thus, preloadinag o f the absorptive cells with fat would allow their isolation by cen- trifugal flotation, thereby, enriching and retaining the goblet cells in the pellet. Such a method of isolation of purified villus cells has already been described by Yc~usef and Muksis ( 1972), who, however, discarded the pelleted goblet cells.

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HOFFMAN AND KUKSIS 1399

Effect of H~rnlcar~es on Lig9ogenesis Previous work with citsate-dispersed mucosal cells

gave no de~nonstrable nlodulation of lipogenesis following addition of several hormones and phar- macolc~gical agents to the incubation rnediaxm (Shakir et a%. 1978). A hormonal response on the part of the intestinal cells was anticipated 01% the basis of their coialrraon cnabryological origin witln the hepa- tocytcs, which have given stash :a, response in vitro (Capuzzi et aH. 197 1 ) . Despite the improved ccll prc- paration, our data on the incorporation of either [lLC]acetate or [14C]glucose into total lipids also failed to indicate a hornlonal response. Likewise, a lack of response was noted in jejunal segments. In a preliminary experiment (Hoffman, ,4. 6. D. and Kuksis, A.: unpublished results) there was an ap- parent depression of the incorporation of [14C] acetate into sterols, by isolated intestinal cells in the presence of glucagon. A comparable effect has been reported in organ culture of canine jejunal tissue after a 6-11 exposure to glucagon (1Om8M) in vitro (Goodman et al. 1979).

Furtlaermore, the bulk of the radioactivity was incorporated into the fatty acids with both glucose and fr~actose in the medium giving no obvious differential eRect on the rate of lipogenesis. This effect is con- trary to the finding irm the liver, where glucagon causes a greater depression of lipogenesis in fructose- stimulated states (Goodridge 1973). Clearly, the ~r~ucosal cells and indeed the intestinal tissue of the adult rat itself either do not possess the appropriate receptors for insulin and glucagon or mucosal lipogenesis simply is not susceptible to hormonal regulation, at least as indicated by the selected tracers and incubation periods. In this connection it is of interest to note that studies on the development of the cytoplasmic giucocorticoid receptors have shown (Henning et aH. 1975) that they are present at high levels in the intestinal mucosa of the rdt during the first 2 postnatal weeks, with the activity showing much lower levels thereafter. Possibly in the adult rat the intestinal lipogencsis is not regulated by cellular Beeels of cyclic ,4MP as it is in the liver (Capuzzi et ul. 1971 ; Allred and Roelmrig 1972; Goodridge 1 975; Geclen et a%. 1978) and in adipose tissue (Goodridge 1968 ) .

In view of the present findings it may be necessary to revise the metabolic interpretation of certain in viva effects of hormones upon the intestinal mucosa of the adult rat. Thus the decreased fat absorption seen in adrenalectomy (Verzar and Laszt 1935) may also be due in part to a suppression of glycoprotein synthesis resulting from dccreasc in the concentration of catecholamines and concomitant mucosd

cellular cyclic AMP levels accornparnying the loss of medullary Inorinones characteristic of this condition (Levin 1969), as wcll as the decreased lipid synthesis proposed previously (&'atson and hffurray 1966; Rodgers c>f a%. 1967). This interpretation of the present results concerning the regulation of glycoprotein synthesis necessary for transport of chylo- microns from the intestine is consistent with the well- known effects of cyclic ,4MP levels on the transport phenomena in the intestine as reviewed by Sernka (1 974).

In conclusion, the increased glycoprotein s p - thesis and secretion observed in rcsponse to hormonal stirnulati011 indicates that the enzyme dispersion of mucosal cells is compatitde with the retention of the receptors for at least the adrenerglc hormones and (or) the capability of a physiological response with complex metabolic and secretory events.

Acknowledgments The authors wish to express their tlaanks to Dr.

B. 6. Louis sf the Department of Obstetrics and Gynecology, University sf Toronto, for useful dis- cussions and helpful criticisms. This work was sup- ported by a grant from the Medical Research Council of Canada (MT-1846). Thc Council also provided a traineeship to A. G. Do Hoffman.

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In vitro response of enzyme-dispersed rat intestinal villus and crypt cells to hormones

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