A MODEL FOR THE "PERMISSIVE" EFFECT OF GLUCOCORTICOIDS ON THE GLUCAGON INDUCTION OF AMINO ACID TRANSPORT IN CULTURED HEPATOCYTES

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A MODEL FOR THE "PERMISSIVE" EFFECT OF GLUCOCORTICOIDS ON THE GLUCAGON INDUCTION OF AMINO ACID TRANSPORT

IN CULTURED HEPATOCYTES

MICHAEL W. ΡARIZ A ROLF F. KLETZIEN2

VAN R. POTTER

McArdle Laboratory for Cancer Research Medical School

University of Wisconsin Madison, Wisconsin

I. INTRODUCTION

The ability of glucocorticoids to potentiate cyclic AMP (cyclic adenosine 3', 5¹-monophosphate) mediated processes has been termed "permissive" (Ingle, 1952; Thompson and Lippman, 1974; Kletzien et al., 1975). However, the biochemical phenomena involved in glucocorticoid "permissive" effects are poorly under-

^-Present address: Department of Food Microbiology and Toxi- cology, Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706

2Present address: Sidney Färber Cancer Center, 35 Binney Street, Boston, Massachusetts 02115

379

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380 M I C H A E L W . P A R I Z A et al

stood. Schaeffer et al. (1969) reported that one site of gluco- corticoid "permissive" action on glycogenolysis was to increase the pool of inactive liver glycogen Phosphorylase, and Butcher et al. (1971) found that long-term exposure of H-35 hepatoma cells to hydrocortisone increased the basal level of tyrosine amino- transferase (TAT) activity as well as TAT induction by dibutyryl cyclic AMP. Zahlten (1974) has commented that glucocorticoids could induce many enzymes involved in cyclic AMP-mediated pro- cesses.

We recently reported (Kletzien et al.^r 1975) the identifica- tion of an amino acid transport system as a site of glucocorticoid

"permissive" action in cultured hepatocytes at the membrane level.

This finding may be of fundamental importance in the process of gluconeogenesis from amino acids. During periods of prolonged fasting, gluconeogenesis by liver and kidney cells from amino acids (especially alanine) mobilized from muscle protein (Exton, 1972, but see also Odessey et al., 1974) is the principal

mechanism whereby normal blood glucose levels are maintained.

Additionally, Park (1974) has commented that in vivo the concen- tration of amino acids is rate-limiting for their conversion to glucose. Thus, our finding that glucocorticoids interact with glucagon in the regulation of the transport of substrates for glu- coneogenesis has important implications.

II. MATERIALS AND METHODS

Figure 1 summarizes the methods and procedures which we use to prepare non-proliferating monolayer cultures of adult rat liv- er parenchymal cells. Methods for determining amino acid trans- port in cultured hepatocytes using the non-metabolizable alanine analog, α-aminoisobutyric acid (AIB), have also been previously reported (Kletzien et al., 1976a).

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A M O D E L F O R G L U C O C O R T I C O I D S 381

Isolate cells using a modification of the collagenase

Suspend cells in a modified Waymouth's perfusion technique

of Berry and Friend.

Yield: 4 0 0 - 6 0 0 X

MB752/1 medium (0.75-1.0 X 106

cells/ml) 106 cells/liver

Change medium after 3-4hrs

Inoculate cells into plastic culture dishes coated with a thin layer of rat-tail collagen ( ~ 5 0 % of the cells attach within 3-4 hrs)

FIGURE 1 Procedure for isolating and culturing liver paren- chymal cells from adult rats (Berry and Friend, 1969; Bonney et al., 1973; Pariza et al., 1974, 1975, 1976a; Kletzien et al., 1976a).

III. RESULTS AND DISCUSSION

A. Characteristics of Hepatocytes in Primary Monolayer Culture Previously we have shown that isolated rat liver parenchymal cells placed in primary monolayer culture do not degenerate but rather improve biochemically during the first 48 hours in culture

(Pariza et al., 1974, 1975; Kletzien et al., 1976a. Additionally, we have emphasized the desirability of following more than one criterion for viability, and in this regard autoradiography with

3H-leucine has proven particularly useful in estimating the frac- tion of viable cells present in monolayer cultures (Pariza et al., 1975). Autoradiograms prepared with labeled precursors of RNA

(such as %-cytidine) would also be highly relevant. However, cultured hepatocytes do not proliferate in serum-containing media currently in use (Bohney et al., 1974; Pariza et al., 1974, 1975) and for this reason autoradiography with labeled precursors of DNA is not available as a marker for viability of cultured hepato- cytes. This particular property is considered to be significant, because liver parenchymal cells do not divide in the adult rat under normal circumstances. Thus, retention of this property by

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382 M I C H A E L W . P A R I Z A et al.

liver parenchymal cells in culture is to be expected if the adult differentiated state is to be maintained (Bissell et al., 1973;

Bonney et al., 1974; Pariza et al., 1975).

There are certain undeniable difficulties which accompany any attempt to study "normal" cells in culture. This is particularly true when the cells under study are from an adult tissue and are therefore highly differentiated. We have discussed many of these problems in previous reports as well as our attempts to determine through biochemical analysis exactly how "adult" non-proliferat- ing cultured hepatocytes really are (Pariza et al., 1974, 1975).

While one cannot expect these cells to perform every liver cell function at once (Pariza et al., 1974), it is nonetheless true that the primary culture system offers many advantages not avail- able with other systems for studying liver. One of these advan- tages is the ability to study the effect of hormones added singly, simultaneously or sequentially using a large number of cultures simultaneously prepared from the liver of one rat (Pariza et al., 1976b). Another advantage is the simplicity with which membrane transport systems can be studied (Kletzien et al., 1976a,b).

B. Hormonal Induction of Amino Acid Transport

Table I shows the effect of glucagon, dibutyryl cyclic AMP, dexamethasone, and insulin, alone and in combination, on the transport of AIB in adult rat liver parenchymal cells in primary culture. Glucagon, insulin and dibutyryl cyclic AMP stimulated the transport of AIB whereas dexamethasone (a synthetic gluco- corticoid) alone or in combination with insulin has no affect on AIB transport. However, treatment with dexamethasone in combina- tion with glucagon or dibutyryl cyclic AMP resulted in a 2-fold stimulation of AIB transport over that observed with either glu- cagon or dibutyryl cyclic AMP alone (Table I ) . This is to our knowledge the first published demonstration of a glucocorticoid

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A MODEL FOR GLUCOCORTICOIDS 383 TABLE I The Effect of Glucagon, Dibutyryl Cyclic AMP,

Dexamethasone, and Insulin Alone and in Combination

on á-aminoisobutyric Acid (AIB) Transport in Cultured Hepatocytes CKletzien et al., 1975; Kletzien et al., 1976a)

AIB transport

Hormone (n moles/4 minutes/mg protein)

None 0.71 ⁺0.02

Dexamethasone 0.70 ⁺0.03

Glucagon 1.33 ⁺0.06

Dexamethasone and glucagon 2.77 ^±0.11 Dibutyryl cyclic AMP 1.66 ^±0.06 Dexamethasone and dibutyryl 3.43 ⁺0.10

cyclic AMP

Insulin 1.54 ⁺0.05

Dexamethasone and insulin 1.55 ⁺0.05

"permissive" effect on an amino acid transport system.

Extensive characterization of the "permissive" action of glucocorticoids on the glucagon induction of amino acid transport in cultured hepatocytes is in progress and will be published sepa- rately (Pariza et ai., 1976a; Kletzien et al., 1976b). However, we would like to present in this communication a summary of relevant findings to date.

1. The glucocorticoids dexamethasone, hydrocortisone, and corticosterone all produce a "permissive" effect on the glucagon induction of AIB transport.

2. Glucocorticoids increase the Vm a x without affecting the Kj^ of the glucagon-induced transport system.

3. A "permissive" effect is observed when glucocorticoids are given simultaneously with glucagon or before glucagon addition (glucocorticoid added, removed, and then glucagon added).

However, in the latter case the "permissive" response appears af-

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384 MICHAEL W. PARIZA et ai

fected by the rate at which the glucocorticoid is metabolized as dexamethasone gives a more prolonged effect than does hydrocortisone.

4. Cyclic AMP levels increase by more than 50-fold within 10-12 minutes after glucagon addition, and this peak value is followed by a slow decline in cyclic AMP levels. The cyclic AMP response to glucagon appears unaffected by prior exposure to dexamethasone, thus supporting the data of Table I that the glucocorticoid "permissive" effect is beyond cyclic AMP.

5. There is a "lag period" of 1-2 hrs following glucagon addition before the initial increase in AIB transport is detected.

Moreover, exposure to glucagon for only 30 minutes results in a marked induction in AIB transport when assayed at 120, 240 or 360 minutes after glucagon removal. Additionally, a "permissive" effect on transport is seen in cells pretreated with glucocorticoid and then handled as above.

6. In contrast to the results presented above, a "permissive"

transport effect fully induced by treatment with dexamethasone and glucagon together for 12 hrs decays with a half-life of about 60 minutes when glucagon is removed, whether or not dexamethasone is removed simultaneously. Moreover, the half-life of a fully induced "permissive" transport system in glucagon alone (dexamethasone removed) is several hours.

7. Cycloheximide or puromycin block decay of a fully induced transport system when dexamethasone and glucagon are removed.

A model consistent with our findings is shown in Fig. 2. The basic tenet of the model is the existence of a transport system in an inactive form which can be converted to an active form by a process initiated by glucagon. While there is circumstantial evidence that cyclic AMP may be involved in this process since

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A MODEL FOR GLUCOCORTICOIDS 385

Glucagon

second \ nessenger ) -

cAMP? /

Induction

mRNA? Glucocorticoid Inactive

Transport S y s t e m

Stimulates

translation?

assembly?

activation?

transport to membrane?

translation?

assembly?

activation?

transport to membrane?

A c t i v e Transport

cycloheximide / glucagon

S y s t e m / 4 *

Blocks Blocks

Degradation or Inactivation

FIGURE 2

DBcAMP acts in the absence of glucagon (Table I), we do not yet have unequivocal data on this point. Tolbert et al. (1973) re- ported a lack of correlation between cyclic AMP accumulation and gluconeogenesis in suspensions of isolated liver cells treated with catecholamines and Michell (1975) has proposed a role for phosphatidylinositol as a "second messenger" independent of cyclic AMP. It is clear that the mechanism whereby the inactive transport system is converted to an active transport system (Fig.

2) is dependent upon the nature of the glucagon-indicated process, and therefore we can not yet stipulate how this conversion occurs.

However, some possibilities which could be involved are listed in Fig. 2: translation of mRNA, assembly of inactive sub-units, activation of a critical sub-unit, or transport to the "proper spot" in the membrane.

The model (Fig. 2) further proposes that glucocorticoids do not directly interact with the glucagon-initiated process, but rather increase the pool of inactive transport system, possibly through induction of mRNA synthesis. This would explain why glucocorticoids alone do not increase AIB transport, and why they cannot alone support a fully-induced transport system.

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386 MICHAEL W. PARIZA et al.

Our model (Fig. 2) also includes a mechanism for degradation or inactivation of the active transport system. There is strong evidence that cycloheximide or puromycin inhibits or retards this mechanism (Pariza et al., 1976a). Moreover, the data conform to the proposition that the mechanism for inactivation or degradation must itself be induced: short exposure to glucagon (30 minutes) permits the appearance of an active transport system at later time points in cells pre-incubated in dexamethasone or no hormones, whereas removal of glucagon from cells pre-treated 12 hours with glucagon plus hydrocortisone results in a rapid decay of the active transport system.

The model shown in Fig. 2 is at present a working hypothesis.

We propose it to convey our current thinking concerning the pre- sently available data (Potter, 1964). Elsewhere (Pariza et al., 1976b) we have discussed how the "permissive" effect of glucocorticoids on the glucagon induction of amino acid transport in liver in vivo may depend upon a sequence of hormones modulated independently by external physiological stimuli. While it is very difficult to investigate complex hormonal interactions in the whole animal, rat liver parenchymal cells in primary monolayer culture are well-suited for such studies.

ACKNOWLEDGMENTS

The authors are grateful to Miss Joyce Becker and staff for cell culture media and facilities, and to Jon Shaw and Mark Kleinschmidt for technical assistance.

Financial support was provided in part by Training Grant T01- CA-5002 and Grants CA-07175 and R01-CA-17334 from the National Cancer Institute.

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