CHAPTER 8
Study of the effects of hormones
8.1. Hormonul responses exhibited by isolated hepatocytes Hepatocytes respond to a variety of different hormones when incubated in suspension. Rapid responses, involving changes in the concentration of intracellular messengers (e.g. cyclic AMP, inositol trisphosphate, Caz+and diacylglycerol), the phosphorylation of target proteins or the formation of an allosteric regulator, are induced by glucagon, a- and 6-adrenergic agonists, vasopressin and angiotensin I1 (Table 8. I ) . Longer term actions of these hormones or intracellular messengers, which can lead to an increase in synthesis of specific proteins within about 90 min (e.g. the induction of phosphoenolpyruvate carboxykinase activity by cyclic AMP), are also observed in suspensions of isolated hepatocytes. The available evidence suggests that glucagon, adrenaline, vasopressin and angiotension 11 each produce responses in isolated hepatocytes that are qualitatively and quantitatively similar to those induced in perfused liver. This comparison is difficult to make because the use of isolated hepatocytes has allowed the performance of a large number of experiments, directed towards investigation of the mechanism of action of hormones on liver cells, that cannot be carried out using the perfused liver.
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During the past 15 years many workers have studied the hormonal responses of hepatocytes to vasopressin and angiotensin 11. These hormones probably do not normally exert physiological effects on hepatocytes, although they may do so in certain pathological states. They have been particularly useful in studies of the role of inositol 1,4,5-trisphosphate and Ca2+ as intracellular messengers in hepatocytes because they do not induce changes in cyclic AMP. By contrast, adrenaline and other adrenergic agonists can bind to both a- and 0-adrenergic receptors and cause changes in Ca2+as well as cyclic AMP. Many of the actions of insulin and epidermal growth factor (EGF) that are rapid in onset are observed in isolated hepatocytes. These hormones interact with plasma membrane receptors possessing a proteintyrosine kinase catalytic site. The effects of insulin and EGF mainly involve changes in intracellular messengers such as cyclic AMP or Ca2+,and bring about immediate effects on metabolism. For example, the presence of insulin prevents the stimulation of hepatic glycogenolysis by glucagon or adrenaline (Table 8.1). Some slow-onset effects of insulin, such as the inhibition of protein degradation, are also observed in hepatocytes (Table 8.1). Many of the other slow-onset effects of insulin on the liver, observed in vivo, have not been clearly defined in any in vitro system including isolated hepatocytes. Responses of the liver to glucocorticoids are difficult to observe in isolated hepatocytes. This is principally because all these responses are slow in onset and therefore require incubation of the cells for at least 5 h. However, some effects of glucocorticoids on enzyme induction (see Chapter 10) have been observed (Table 8.1).
8.2. Optimal conditionsfor observing the effects of hormones 8.2.1. Isolation of hepatocytes
In early studies of hormone-responsive hepatocytes, some evidence for a loss of hormone receptors was reported (Garrison and Haynes,
TABLE 8. I Examples of responses in rat hepatocytes induced by hormones
Hormone
Example of response
References
Glucagon
Increased gluconeogenesis
Johnson et al. (1972) Claus et al. (1975) Pilkis et al. (1975) Garrison and Haynes (1975) Garrison and Haynes (1975) Pilkis et al. (1975)
Increased 0-utilization Increased pyruvate carboxylation Increased formation of cyclic AMP Increased glycogen synthesis Decreased glucagon- or a,-adrenergic agonist-stimulated glycogenolysis Decreased protein degradation
Johnson et al. (1972) Thomas and Williamson (1983)
Epidermal growth factor
Increased formation of inositol trisphosphate
Johnson and Garrison (1977)
Adrenaline (P-adrenergic receptors)
Increased glycogenolysis Increased formation of cyclic AMP
Cherrington et al. (1977)
Adrenaline (a-adrenergic receptors)
Increased glycogenolysis Decreased glycogen synthesis Increased intracellular free Ca”
Hutson et al. (1976) Hutson et al. (1976) Charest et al. (1985)
Vasopressin
Increased intracellular free Ca”
Increased formation of diacylglycerol
Thomas et al. (1984) Charest et al.. (1985) Thomas et al. (1984) Burgess et al. (1984) Hughes et al. (1984)
Induction of phosphoenolpyruvate carboxykinase
Salavert and lynedjian (1982) Watford et al. (1983)
Insulin
Increased formation of inositol trisphosphate
Blackmore et al. (1979) Hopgood et al. (1987)
Morgan et al. (1983)
0 5 Po
m
7 R 2 2
0 ;a
B2
2 !
h)
Glucocorticoids
8
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ISOLATED HEPATOCYTES
1973). This may have been due to the action of collagenase during perfusion of the liver and to proteases carried over into the final cell suspension (Pilkis et al., 1975). Some workers have used the stimulation by glucagon of gluconeogenesis as a monitor of hormoneresponsiveness and metabolic integrity (Pilkis et al., 1975). These and other studies established that in the preparation of hormone-sensitive cells, hyaluronidase should be omitted from the digestion medium and particular attention paid to: (1) the use of the shortest exposure of the liver to collagenase consistent with the separation of individual hepatocytes; (2) avoidance of hypoxia during perfusion of the liver; and (3) good temperature control of the perfused liver (Zahlten and Stratman, 1974; Pilkis et al., 1975; Hutson et al., 1976; Blackmore and Exton, 1985).These precautions appear to reduce damage to plasma membrane receptor proteins and other proteins involved in intracellular signalling, such as ion channels and transporters, adenylate cyclase and cyclic AMP phosphodiesterase. There is some evidence indicating that, in the preparation of hormone-sensitive hepatocytes from fed rats, the maintenance of high concentrations of glycogen during the isolation procedure improves the actions of glucagon and insulin (Wagle, 1974; 1975). Exton and his colleagues have routinely incorporated washed erythrocytes in the preparation of hormone-sensitive hepatocytes (Hutson et al., 1976; Blackmore and Exton, 1985) in the belief that this improves the quality of the hepatocytes. However, good hormonesensitive hepatocytes can be prepared without including red blood cells in the liver perfusion medium. For most experiments, the additional time and work involved in the use of erythrocytes is probably not warranted. Likewise, the inclusion of 1.5% gelatin in the medium used for incubation and washing of the cells during isolation (Blackmore and Exton, 1983) and the inclusion of fumarate, glutamate, glucose and pyruvate in the perfusion medium (Zahlten and Stratman, 1974) may enhance the quality of the cells, but their inclusion in the experimental protocol is generally considered not to be necessary.
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8.2.2. Incubation medium
An additional buffer is sometimes added to the Krebs-Henseleit medium in order to maintain pH at 7.4 duringthe incubation medium. 2 4 [ 2-hydroxy- 1,1 -bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid (TES) is most commonly used. For example, Haynes and his coworkers (Sistare and Haynes, 1985)have employed 100 mM TES buffer (pH 7.4), and have compensated for the increase in osmolality by decreasing the concentration of NaCI. For the incubation of hepatocytes in hormone studies, we recommend a medium containing 117 mM NaCl; 4.7 mM KCl; 1.2 mM KH,PO,; 1.3 mM Ca2+; 1.2 mM MgSO,; 24 mM NaHCO,; and 20 mM TES, equilibrated with carbogen a t 37°C and adjusted to pH 7.4 with KOH. This is a modification of Krebs-Henseleit bicarbonate buffer (KHBT). Some workers include gelatin or albumin in the incubation medium when studying the actions of hormones such as glucagon. Hutson et al. (1976) used 1.5% gelatin, while Thomas and Williamson (1983) employed 2% dialysed BSA. It has been suggested that gelatin or albumin improve the quality of hepatocytes in suspensions incubated for periods greater than an hour, although there is little definitive evidence about this. In the author’s experience, inclusion of gelatin or albumin for short incubation periods is not necessary, but the use of gelatin, or complex media (e.g. Waymouth medium) is desirable for prolonged incubations. Different approaches have been made to the question of the concentration of CaZ+to be employed in the extracellular incubation medium. There are a few studies which suggest that the presence of physiological Ca*+concentrations in the extracellular medium is actually detrimental to isolated hepatocytes. However, the great majority of workers believe that optimal incubation conditions include the presence of extracellular free Ca2+at a physiological concentration. As discussed earlier (Chapter 2), it is considered desirable to add the Ca2+back to hepatocytes as soon as possible after isolation of the
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cells. It should be noted that the normal concentration of free Ca2+ in the blood of an animal is about 1.3 mM. Therefore, unless a Ca2+-bindingprotein such as albumin is present in the incubation medium, the concentration of total Ca2+in the incubation medium should be 1.3 mM. A pre-incubation at 37°C should be carried out for at least 15 min prior to hormone addition in order to allow re-establishment of basal metabolism and ion gradients. Some workers use an even longer preincubation period. When hormone additions are made to incubation mixtures, the hormones are generally dissolved in 150 mM NaCl to minimize effects on the osmolality of the suspension. In hepatocytes isolated from fed rats, glycogen is slowly broken down during subsequent incubation of the cells. Glycogen stores can be maintained by inclusion of glucose at a concentration of 15 mM in the incubation medium. Extracellular glucose both maintains adequate stores of glycogen, which may be required in studies of glycogen metabolism, and provides an exogenous source of energy for the cell. 8.2.3. Incubation vessel
In practice, the most convenient incubation vessel is a 20-ml plastic screw-capped scintillation vial. Cell incubations can also be successfully performed in 25-ml or 250-ml Erlenmyer flasks. In each case the volume of the incubation medium should be about 10% of the total volume of the incubation vessel. This ratio of incubation medium to gas space allows adequate oxygenation of cells. In all cases the containers should be gassed with carbogen and shaken at 90 oscillationdmin. The concentration of cells should be within the range 1-5 x lo6 cells/ml. There is a risk of hypoxia if a higher concentration of cells is used as the rate of diffusion of O2 into the cells may become limiting. A disadvantage of the incubation vessels described above is that they are not convenient for rapid removal of samples from the incubation medium. This difficulty can be overcome by the use of stationary
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cylindrical water-jacketed incubation chambers (diameter 2 cm, height 6 cm) in which the incubation medium is stirred. The incubation module designed by Yellow Springs Instruments for measurement of 0,-tension (Section 6.5.1) is highly satisfactory (Barritt et al., 1981). Four or eight of these cylindrical incubation chambers (one or two modules) can be employed at one time. The use of these chambers allows a large number of samples, ranging in volume from 0.1-2.0 ml, to be removed from the incubation mixture at relatively short time intervals. Thus it is possible to obtain time courses for the effects of hormones on enzyme activities such as glycogen phosphorylase and on the level of intracellular messengers (e.g. cyclic AMP). Hepatocytes can be incubated satisfactorily for periods up to 60 or 90 min using the stirred cylindrical chambers. However, the cells exhibit higher rates of leakage of LDH than cells incubated in shaken plastic vials.
Protocol 8.1 Effect of glucagon on cyclic A M P concentrations in hepatocytes (i) After their preparation, suspend the hepatocytes at a density of 6 x lo6 cells/ml in KHBT medium at a final pH of 7.4. (ii) Set up a number of incubation vials as follows. Add the cell suspension (0.5 ml) to 1.O ml of KHBT-medium in a 20-ml plastic vial and gas the vial for at least 10 s with carbogen at 1 I/min and seal the vial. Incubate the cells for 15 min. Add glucagon M will give maximum response) and continue incubation for the desired period of time. At the selected times (prior to and after the addition of glucagon) remove duplicate samples of the cell suspension (0.5 ml), and mix each sample with 0.6 M PCA (0.4 ml). After centrifugation at 1000 x g , in order to rerpove precipitated protein, and adjustment of the pH to 7.0 (Protocol 6.2), determine the amount of cyclic AMP present. Cyclic AMP is commonly measured by a protein binding assay (Gilman, 1970). The incubation conditions described in Protocol 8.1 can also be
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used in studying the effects of glucagon on enzyme induction, for example the induction of phosphoenolpyruvate carboxykinase. However, the incubation period should be no longer than 90 min. For longer incubations, the conditions of Protocol 8.3 are used.
8.3. Special conditions for the study of specific hormonal effects Changes in the concentrations of phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5-trisphosphate or diacylglycerol, as a consequence of hormone action, have been investigated by employing a variety of radiolabelled compounds. Workers have studied hormonal effects on the following reactions: phosphatidylinositol 4,5-bisphosphate hydrolysis (using ['Hlinositol and "Pi); inositol polyphosphate formation ([-'H]inositol); phosphatidyl choline hydrolysis and diacylglycerol formation (['Hlcholine, ['Hlglycerol and [3H]arachidonic acid). Cells have been successfully labelled with 32Pi(Billah and Michell, 1979; Kirk et al., 1979; Tolbert et al., 1980; Litosch et al., 1983; Seyfred and Wells, 1984), pH]inositol (Tolbert et al., 1980; Prpic et al., 1982; Litosch et al., 1983; Johnson and Garrison, 1987) or [-'H]arachidonic acid (Takenawa et al., 1982; Thomas et al., 1983; Pickford et al., 1987) by incubating hepatocytes in the presence of the labelled compound. An example of the pre-labelling technique is given below.
Protocol 8.2 The pre-labelling of hepatocytes with [JH]inositol (i) After isolation, suspend the hepatocytes at a density of 6 x lo6 cells/ml in KHBT medium containing 1.5% gelatin (KHBT/gelatin) and keep the cells at 37°C. (ii) Add the hepatocyte suspension (0.75 ml) to 1.25 ml of KHBTIgelatin in a 20-ml plastic vial. Add glucose to give a final con-
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centration of 15 mM, and my0-[2-~H]inositol (40 Ci/mol) to give a final concentration of 0.1 mM. After addition of the hepatocyte suspension, gas each vial for 10 s with carbogen delivered at a rate of 1 I/min and seal the vial. Incubate the cell suspensions at 37°C and shake at 90-120 oscillations per min. The total incubation time is 60 min. Gas each vial as described, every 30 min. After completion of the incubation, centrifuge the cell suspension at 50 x g for 75 s, remove the supernatant and suspend the cells in 2 ml of fresh KHBT/gelatin. Repeat this washing step once. The final cell suspension (about 2 x loh cells/ml) can then be incubated under the desired conditions in the presence or absence of the hormone under test. The cells respond quite well to hormones after the pre-labelling period. The labelling of isolated hepatocytes by the method described in Protocol 8.2 is much less expensive than a frequently employed alternative procedure that involves the administration of the radioactive label to rats by intraperitoneal injection (Kirk et al., 1979; Prpic et al., 1982; Polverino and Barritt, 1988). However, the labelling of hepatocytes with some compounds, such as [3H]choline o r ['Hlglycerol, requires incubation times longer than an hour, especially if an approach to steady-state, with respect to the distribution of the label, is required. When using these labelled metabolites it is therefore desirable to administer the radiolabelled compound to rats by intraperitoneal injection.
8.4. Actions of insulin Although hepatocytes respond to insulin (Table 8.1) the concentrations of the hormone required to induce changes in hepatocyte metabolism are often higher than those required to induce effects in the perfused liver (Pilkis et al., 1975). Moreover, the effects of insulin on hepatocytes are somewhat variable. It seems that insulin receptors, and receptors for other hormones and growth factors in which the
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receptor protein includes a tyrosine kinase catalytic site, are more susceptible to damage during isolation of the hepatocytes. However, this point is not well documented. Hopgood et al. (1977) described specific conditions for the study of the inhibition by insulin of protein degradation in rat hepatocytes over a period of 6 h. Maximal proteolysis and response to insulin were obtained in an incubation mixture consisting of Ca*+-free KHBTlgelatin that also contained 2 mM leucine, 16.5 mM glucose, 20 mM TES, 100 pM EDTA and 10 pglml phenol red, 60 pglml of penicillin and 10 pg/ml of streptomycin sulphate. The incubation vessels were polyethylene scintillation vials with a volume of 20 ml. Each vial contained 2-3 x lo6 cells in a total volume of 1.5 ml. The authors suggested that the function of EDTA was to chelate heavy metal ions; EDTA appeared to protect cells from damage, while the omission of Ca2+reduced clumping.
8.5. Action of steroid hormones The induction of enzymes such as phosphoenolpyruvate carboxykinase and tyrosine aminotransferase by glucocorticoids is slow in onset. This presents a problem in studies with isolated hepatocytes since relatively long periods of incubation are required. A successful protocol for investigation of the effects of glucocorticoids on the induction of tyrosine aminotransferase activity that can also be used for studies of enzyme induction by non-steroid hormones, such as glucagon, is given in Protocol 8.3.
Protocol 8.3 Induction by dexamethasone of tyrosine aminotransferase activity (i) At the final stage of preparation suspend the hepatocytes, at a density of between 1 x 106 and 3 x lo6 cells/ml, in Waymouth medium modified to contain 0.2% BSA, 100 U/ml of penicillin and 100 pglml streptomycin.
Ch. 8.
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(ii) Incubations are performed in 20-ml plastic vials. To each vial, add 1.7 ml of the cell suspension and 0.3 ml of 5 pM dexamethasone in 0.9% NaC1. Gas the cell suspension with carbogen for 10 s and seal the tubes at the beginning of the incubation. Repeat gassing procedure at 30-min intervals during the incubation. The vials are incubated at 37°C with reciprocal shaking at 90 cycles per min. On completion of the incubation period of 5 h, remove a sample of the cells (1.0 ml) for assay of tyrosine aminotransferase activity. Since the incubation period is relatively long there is some degree of cell damage. Under optimal conditions the number of cells which exclude trypan blue decreases from greater than 90% at the beginning of the incubation to about 75% or 80% after 5 h.
8.6. Hormone effects on mitochondriu isolated from hepatocy tes It is also possible to study changes in mitochondria isolated from hepatocytes that have previously been exposed to a hormone. In the protocol below a Dounce homogenizer is used to rupture the cells. Care must be taken that this procedure does not also damage the mitochondria.
Protocol 8.4 Isolation of intact mitochondria from hepatocytes (i) Incubate hepatocytes (final density 1 x lo6 cells/ml) in 250-ml glass Erlenmyer flasks, shaken at 90 oscillations per min, at 37"C, in a medium comprising KHBT, 100 pM EDTA, 1.5% gelatin and 15-20 mM glucose (for studies on cells from fed rats) and with an atmosphere of carbogen. The total volume of the cell incubation mixture in each flask is 60 ml. In studies of hormone effects on mitochondria, an incubation time of between 0 and 90 min for pre-treatment of the intact hepatocytes with hormone can be used without undue damage to the cells. Add the appropriate quantity of the hormone to each flask to obtain the hormone concentration range under study.
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(ii) Centrifuge the contents of each flask at 50 x g for 45 s at room temperature. Wash each pellet twice with 40 ml of ice-cold 250 mM sucrose, 5 mM HEPES, 0.5 mM EGTA, 0.05‘%,defatted BSA, adjusted to pH 7.4 at M ” C with KOH (Isolation Medium plus EGTA). Suspend each cell pellet (about 1 g wet weight of loosely packed cells) in 6 ml of Isolation Medium plus EGTA. (iii) Carry out the following steps at 0 4 ° C . Homogenize each cell suspension in a glass Dounce homogenizer (7 ml capacity) with 30 upand-down strokes of a tight-fitting glass pestle (Wheaton “A” or Kontes “B”). Centrifuge the homogenate at 300 x g for 10 min and remove the supernatant. Centrifuge the supernatant at 4500 x g for 5 min. The resulting pellet is composed mainly of mitochondria. Resuspend the mitochondrial pellet in 5 ml of Isolation Medium, free of EGTA, in the homogenizer by means of two up-and-down strokes of a loose-fitting pestle (Wheaton “B” or Kontes “A”). Pour into a centrifuge tube, add a further 4 ml of EGTA-free Isolation Medium, mix and centrifuge at 4500 x g for 5 min. Suspend the resulting mitochondrial pellet in 0.45 ml of Isolation Medium, without EGTA, and keep at U ” C . The expected yield of mitochondria is about 10 mg mitochondrial protein from 1.0 g wet weight of cells. The respiratory control ratios (the ratio of the rate of 0, consumption in the presence of ADP compared with that after conversion of all ADP to ATP), measured in the presence of succinate, should be about 4.5 for mitochondria from freshly-isolated hepatocytes, and 3.5 for mitochondria from hepatocytes previously incubated at 37°C for 60 min, compared with a value of about 6.0 for mitochondria prepared from fresh liver tissue. The presence of defatted BSA in the media used for the isolation and washing of the mitochondria is important as this improves their quality. This is reflected in a significant increase in ADP-stimulated respiration and in the respiratory control ratio. There is some variation in the tightness of fit of different Dounce homogenizers. Therefore a new homogenizer should be tested to deter-
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mine the number of passes required to optimize the yield and quality of mitochondria. Unless considerable cure is exercisrd thr Doirricc homogenizer can damage the mitochondria. An important factor in the isolation of well-coupled mitochondria from hepatocytes is the integrity of the hepatocytes during incubation at 37°C. Good mitochondria are obtained from cells which have suffered least damage. I t is considered that the inclusion of a protein such as albumin or gelatin (see Chapter 6 ) and maintenance of the pH of the incubation medium at 7.4 play an important par1 i n improving the quality of the cells at the end of the incubation period. Gellerfors and Nelson ( 1979) have used sonication to break hepatocytes, and have obtained well-coupled mitochondria. However, in our laboratory it has proven difficult to find conditions of sonication which give a high yield of well-coupled mitochondria (Hughes and Barritt, unpublished results). The time of sonication (about 15 s) recommended by Gellefors and Nelson is very short, making it difficult to avoid under- or over-sonication of the cells. Shears and Kirk (1984a) have developed a method for the preparation of small quantities of a mitochondria-enriched fraction from isolated hepatocytes. The method involves lysis of the cells with digitonin, mechanical disruption of the cells by forcing the cell suspension through a 23G syringe needle at high pressure, followed by rapid centrifugation of the broken cells through a layer of silicone oil. This results in a partially-purified mitochondria1 fraction which is contaminated with cell nuclei. This method has been used to measure mitochondrial membrane potential (Shears and Kirk, 1984a) and the amount of 45Ca2+in mitochondria from hepatocytes treated with hormones (Shears and Kirk, 1984b). Measurements of rates of respiration, Ca" movement and other metabolic parameters have shown that the properties of mitochondria isolated from hepatocytes are similar to those isolated from intact tissue, although, as noted above, the respiratory control ratios of mitochondria prepared from cells are lower than those prepared
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from intact liver tissue (Garrison and Haynes, 1975; Hughes and Barritt, 1984). However, there have been few rigourous assessments of the degree of contamination of the mitochondria1 fraction by constituents of other organelles by means of a complete analysis of distribution of marker enzymes.