Intracellular redox state and stimulation of gluconeogenesis and glycogenolysis in isolated hepatocytes from the pig (Sus scrofa)

Comp. Biochem. Physiol. Vol. 69111,pp. 775 to 779, 1981

0305-0491/81/080775-05502.00/0 Copyright © 1981 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

INTRACELLULAR REDOX STATE AND STIMULATION OF GLUCONEOGENESIS AND GLYCOGENOLYSIS IN ISOLATED HEPATOCYTES FROM THE PIG (SUS SCROFA) MICHAEL G. CLARK, IVANG. JARRETTand OWEN H. FILSELL CSIRO Division of Human Nutrition, Kintore Avenue, Adelaide, SA 5000, Australia and ALFONSA. BONDEand ROBERTW. PHILLIPS Department of Physiology and Biophysics, Colorado State University, Fort Collins, CO 80521, USA (Received 29 December 1980) Abstract--1. Isolated pig hepatocytes were prepared and the effects of changes in the cytoplasmic [NADH]/[NAD +] ratio on the efficacy of glucagon to alter rates of metabolism were examined. 2. With hepatocytes from fed pigs 1 #M-glucagon stimulated glucose output. The response to giucagon was similar in magnitude regardless of whether 10 mM-lactate or 10 mM-pyruvate was present in incubations. 3. With hepatocytes from 72-hr fasted pigs, glucagon (1 #M) increased the rate of gluconeogenesis from 10 mM-pyruvate but was without effect on the rate from I0 mM-lactate. These results differed from those obtained using rat hepatocytes where 1 #M-glucagon increased gluconeogenesis from 10 mMlactate and inhibited gluconeogenesis from 10 mM-pyruvate. 4. Intracellular concentrations of lactate and pyruvate were measured following 10min incubations of pig hepatocytes with 10 mM-lactate or 10 mM-pyruvate. Comparisons with similar experiments conducted using rat hepatocytes indicated that both lactate and pyruvate entered the cells of both species and significantlyaltered the lactate/pyruvate ratio. 5. Properties of the membrane-bound low Ks cyclic AMP phosphodiesterase from pig and rat liver were compared. The activity of the enzyme in each species was similar and was inhibited to the same extent by NADH. 6. The inability of pyruvate to inhibit the stimulatory effect of glucagon on glucose output and gluconeogenesis in pig hepatocytes does not appear to result from differences in the permeation of substrate into the cells or the sensitivity of cyclic AMP phosphodiesterase to altered cytoplasmic [NADH]/FNAD +] ratio mediated by pyruvate or lactate addition.

INTRODUCTION We have previously reported that the ability of glucagon to stimulate glucose output (predominantly from glycogen breakdown) by isolated rat hepatocytes and perfused rat liver is affected by the redox state of the cytosolic NAD ÷ couple (Clark et al., 1977). In hepatocytes from fed rats an increase in pyruvate, NH~, or 02 concentration or a decrease in the [lactate]/[pyruvate] or [sorbitol]/[fructose] ratios decreased the ability of 1/~M-glucagon to stimulate glucose output without significantly altering the control rate. A more recent attempt was made to elucidate the mechanism, at the cellular level, by which glucagon responsiveness was altered by changes in redox state (Clark & Jarrett, 1978). These studies suggested that oxidized substrates such as pyruvate altered the redox state of the cell and prevented the accumulation of sufficient cyclic AMP to activate the protein kinase-cascade sequence. Since NADH was found to inhibit the low-K,, cyclic AMP phosphodiesterase, it was proposed that de-inhibition of this enzyme by removal of NADH provided the molecular basis for the effect of redox state on the hormonal control of rat hepatocyte metabolism by glucagon. More recently, the role of 775

the cellular redox state in the hormonal stimulation of gluconeogenesis was studied in hemoglobin-free perfused rat liver, by fluorimetric measurement of the redox states of intracellular pyridine nucleotides (Sugano et al., 1980). Stimulation of gluconeogenesis from lactate by glucagon was affected by the lactate/ pyruvate ratio; a decrease in the lactate/pyruvate ratio resulted in a decrease in the efficacy of glucagon with a decrease in pyridine nucleotide fluorescence. In addition, the remaining stimulation by glucagon of glucose production from pyruvate was completely abolished during octanoate infusion (causing a more reduced mitochondrion) although it was observed when the cytosol was reduced during ethanol infusion. These results suggested that excess NADH in the cytosol is essential for total glucagon efficacy. Various aspects of pig metabolism more closely resemble that of the human than does the rat. Thus attempts have been made to isolate pig hepatocytes for human-related studies (Belfrage et al., 1975; Jarrett et al., 1980). In the course of characterising isolated pig hepatocytes in our laboratories we noted that the effects of altered redox state on glucagon efficacy as seen with rat liver preparations (Zahlten et al., 1973; Rognstad, 1975; Mapes & Harris, 1976;

776

MICHAEL G. CLARK et al.

Clark et al., 1977; Sugano et al., 1980) were not apparent. Thus the present report compares rat and pig hepatocytes in this respect.

MATERIALS AND METHODS Materials

Glucagon (lot 258-V016-235) was a generous gift from Dr W. Bromer, Eli Lilly, Indianapolis, IN, and freshly prepared in 0.9% (w/v) NaCI. L( + )-lactic acid and sodium pyruvate were purchased from Sigma Chemical Co., St. Louis, MO, and were used as freshly prepared aqueous solutions (pH 7.4). Collagenase (type II) was purchased from Worthington Biochemical Corp., Freehold, NJ. DowCorning silicone oil was obtained from Ajax Chemicals, Bankstown, NSW, Australia. Bovine serum albumin (fraction V) was purchased from Calbiochem.. La Jolla, CA. Animals Adult miniature pigs (Hormel strain, body wt 2 ~ 3 0 kg) maintained at Colorado State University were used for part of this study, together with adult feral pigs (42kg approx.) reared at the CSIRO laboratory, Adelaide. Pigs at Colorado State University were fed a custom mixed all purpose diet (Panepinto et al., 1978) and pigs at CSIRO received "Danex" Pig Grower Pellets, Noske Flour Mills Pty. Ltd., Adelaide, SA, Australia). For studies on gluconeogenesis, pigs were starved for 72 hr before cell isolation. Times less than 48 hr did not significantly deplete glycogen concentrations. Male Hooded Wistar rats weighing 200-250 g and either fed or starved for 48 hr (where indicated) were used. Hepatocyte isolation and incubation Rat hepatocytes were prepared essentially as described by Berry & Friend (1969), with the omission of hyaluronidase. Several methods were initially tested for preparing isolated pig hepatocytes. However as indicated by Belfrage et al. (1975) the method involving sequential perfusion with buffers containing 0.5 mM-EGTA then 2 m M Ca 2+ with 0.1~o (w/v)collagenase gave the most efficient dispersion of cells. This latter method is essentially that used by Seglen (1973) for isolating rat hepatocytes. Approximately 40 g of a peripheral portion of a ventral lobe of the liver was excised, cannulated and perfused through a branch of the portal vein in a manner similar to that used for preparing lamb hepatocytes (Clark et al., 1976). Non-circulating perfusion with Ca 2 +-free Krebs improved Ringer I containing pyruvate (Dawson et al., 1969) and 0.5mM-EGTA was started and continued for 8 rain. The perfusion was then changed to a re-circulating mode and continued for 60 rain with Krebs improved Ringer I containing 2 mM Ca 2 ÷ and 0.17o (w/v) collagenase. Flow rate was maintained at approx. 60 ml/min by pumping directly to the liver from a Masterflex peristaltic pump (Cole-Parmer Instrument Co., Chicago, IL). The perfusate was equilibrated at pH 7.4 and pO2 of 400 + 50mm Hg using 02 + CO2 (19:1). At the end of the collagenase perfusion the partially digested liver was teased apart and the suspension of cells filtered through a single layer of terylene mesh (1 mm x 0.4 ram). The liver suspension (60 ml) representing 20-30~o by wt of the lobe was incubated with shaking at 37°C for 15 min and gassed with the 02 + CO2 mixture. At the end of the incubation, the cell suspension was filtered again and washed three times as described for lamb liver cells (Clark et al., 1976). The yield of cells using this procedure was approx. 10~o and within the range reported by Belfrage et al. (1975). The number of cells per g dry wt was 3,7 x 10a and 9.1 × 108 for fed and fasted pigs, respectively. Average viability figures, assessed by trypan blue exclusion, were

also within the range reported by Belfrage et al. (1975) and were 87~o and 78% for fed and fasted pigs, respectively. The two strains of pigs were compared. No significant difference between the properties of the hepatocytes to synthesize glucose was indicated (analysis of variance). On this basis, data from the two laboratories involving both strains have been pooled. Pig and rat hepatocytes were finally suspended in Krebs--Henseleit saline containing 2.5 mM Ca 2 + and 2.5°~.~, (w/v) bovine serum albumin. Analytical determinations Rates of glucose output and gluconeogenesis were determined in stoppered glass vials of 20 ml capacity with shaking (80 oscillations/rain) at 37'~C, with substrate and hormone in a total vol of 1.5 ml of the albumin-containing buffer (2.5 mM Ca 2+ final concentration). After addition of the cells (approx. 13 mg dry wt) each vial was gassed with the 02 + CO2 mixture and stoppered for the entire 30 min incubation. The reactions were stopped by the addition of 0.5 ml of 670 (w/v) HCIO 4. After thorough mixing, precipitated protein was removed by centrifugation (5000 0 for 10 rain), and the supernatant was analysed for glucose content by the glucose oxidase method of Huggett & Nixon (1957) in an autoanalyser [Technicon (Ireland) Ltd., Dublin, Republic of Ireland]. The method for the separation of hepatocytes from medium was based on the silicone-oil centrifugation method of La Noue et al. (1972) as described previously (Clark & Jarrett, 1978). Lactate (Hohorst, 1963) and pyruvate (BiJcher et al., 1963) were determined on neutralized perchlorate extracts of a sample of the lower HC104 layer. Preparation oJ'liverJJ'actions containing cyclic A M P phosphodiesterase activity The method used was essentially that of Loten et al. (1978) as described previously (Clark & Jarrett, 1978) except that fresh whole liver was used in place of isolated hepatocyte suspensions. The low K I cyclic AMP phosphodiesterase (EC 3.1.4.17) was assayed (Arch & Newsholme, 1976) in solubilized membrane fractions using 0.1/~M cyclic AMP.

RESULTS AND DISCUSSION Several laboratories have implicated the involvement of redox state in polypeptide h o r m o n e action in rat liver. Zahlten et al. (1973), G a r r i s o n & Haynes (1973), Rognstad (1975), Clark et al. (1977) and Sugano et al. (1980) have all noted that the response to glucagon by rat liver was dependent upon whether pyruvate or lactate was the gluconeogenic substrate. An effect of altered redox state on glucagon efficacy was also noted when pathways not involving reducing equivalent input or o u t p u t were studied (Clark et al., 1977). Subsequently a detailed examination of the possible mechanism by which oxidized substrates such as pyruvate impaired the response to glucagon in rat hepatocytes led to the proposal that these substrates achieved their effect by lowering the cytosolic [ N A D H ] / [ N A D +] ratio a n d deinhibited the low K,, cyclic A M P phosphodiesterase. T h u s insufficient cyclic A M P was able to accumulate when glucagon activated adenyl cyclase. This proposal was largely supported by the discovery that micromolar levels of N A D H inhibited the cyclic A M P phosphodiesterase (Clark & Jarrett, 1978) solubilized from rat hepatocyte m e m b r a n e fractions. The present data suggest that this proposal m a y not apply in the pig.

Cytoplasmic NAD couple and glucagon efficacy

777

Table 1. Effect of extracellular lactate and pyruvate on glucagon-mediated changes in glucose output by isolated hepatocytes from fed pigs and rats Pig Rate of glucose output (/~mol/ rain per g dry wt) Additions Saline Glucagon 10 mM-Lactate 10 mM-Lactate + glucagon 10 mM-Pyruvate 10 mM-Pyruvate + glucagon

Rat % Increment due to glucagon

3.1 _ 0.4 (9) 5.5 ___0.6 (9) 2.8 + 0.4 (9)

77.4

Rate of glucose output (/zmol/ min per g dry wt) 8.9 + I.I (14) 17.8 -I- 1.2 (14) 10.0 _+ 1.1 (14)

% Increment due to glucagon

100.0

5.3 + 0.7 (9) 3.5 + 0.5 (9)

87.0

19.4 +_ 1.1 (14) 9.3 _ 1.1 (14)

94.0

5.8 + 0.6 (9)

65.7 NS

11.7 + 1.1 (14)

25.8*

Isolated hepatocytes were prepared as described in the Materials and Methods section. In addition to lactate and pyruvate the cells (approx. 13 mg dry wt) were incubated with 1 gM-gtucagon or an equal vol of 0.99/o saline in a total vol of 1.5 ml of buffer for 10 min at which time HCIO4 was added to give a final concentration of 1.5~o (w/v). Glucose output was determined as described in the text. Means + SEM are shown with the numbers of cell preparations in parentheses. An analysis of variance was applied to assess significant differences between the increments due to glucagon in the presence of pyruvate vs lactate;* significant, NS, not significant.

Table 1 shows the effects of extracellular lactate and pyruvate on glucagon-mediated changes in glucose output by isolated pig and rat hepatocytes from fed animals. As reported previously (Clark et al., 1977; Clark & Jarrett, 1978) the efficacy of glucagon to stimulate glucose output (predominantly derived from glycogen breakdown; Clark et al., 1977) by rat hepatocytes was markedly diminished by the inclusion of 10 mM-pyruvate in the incubations. However for pig hepatocytes the efficacy of glucagon to stimulate glucose output was unaltered by the inclusion of pyruvate, thus indicating a major species difference. As noted previously by other workers (Zahlten et al., 1973; Garrison & Haynes, 1973; Sugano et al., 1980) and ourselves (Clark et al., 1977) glucagon stimulated gluconeogenesis in rat hepatocytes (starved animals) from 10 mM-lactate but inhibited or had no effect on gluconeogenesis from 10mM-pyruvate. In contrast to these findings and as shown in Table 2,

glucagon stimulated gluconeogenesis from 1 0 m M pyruvate in pig hepatocytes (starved animals) but was without significant effect on the rate from 10raMlactate. Thus data from both Tables 1 and 2 indicated that a relatively oxidised substrate such as pyruvate had essentially no effect on the efficacy of glucagon to alter metabolism in pig hepatocytes. The next experiment was thus conducted to assess whether differences existed between the species for the rates of lactate and pyruvate entry into cells. Table 3 shows the effect of incubating cells with lactate or pyruvate on the intracellular concentrations of lactate and pyruvate and on the lactate/pyruvate ratio. The intracellular concentrations of lactate and pyruvate in rat liver cells incubated alone (with neither lactate nor pyruvate) were 1.37 and 0.087 prnol/g wet wt, respectively; these compared favourably with value for whole liver (Williamson et al., 1967). The corresponding concentration of lactate and pyruvate in p i g liver cells was approx. 259/o of those of the rat, but the lactate/

Table 2. Effects of substrate and glucagon on gluconeogenesis in isolated hepatocytes from fasted pigs and rats

Saline Substrates Endogenous 10mMLactate 10mMPyruvate

Rate of gluconeogenesis (/~mol/min per g dry wt) Pig Rat Glucagon Saline Gtucagon

0.46 _+ 0.17 (4)

0.59 ___0.15 (4)(NS)

0.44 _+ 0.28 (4)

0.59 ___0.33 (4)(NS)

1.28 _+ 0.35 (6)

1.46 _+ 0.39 (6)(NS)

1.55 _+ 0.33 (4)

2.27 _+ 0.37 (4)(P < 0.05)

1.69 _+ 0.25 (6)

2.00 +_ 0.26 (6)(P < 0.01)

2.90 __ 0.46 (4)

1.95 4- 0.49 (4)(P < 0.01)

Isolated hepatocytes were prepared as described in the Materials and Methods section. In addition to the substrates shown, the cells (approx. 13 mg dry wt.) were incubated with 1 #M-glucagon, or an equal vol of 0.9% saline in a total vol of 1.5 ml of buffer for 30 min at which time HCIO4 was added to give a final concentration of 1.5% (w/v). Glucose was determined as described in the text. Means _+ SEM are shown with the numbers of cell preparations in parentheses. A paired t-test was applied to assess significant differences when compared with the corresponding control (saline) value; NS, not significant.

778

MICHAEL G. CLARK et al, Table 3. Effect of added lactate and pyruvate on the intracellular concentration of lactate and pyruvate in isolated hepatocytes from pigs and rats

lntracellular metabolite concentration (l~mol/g wet wt of cells) Pig Rat Lactate/ Lactate/ Lactate Pyruvate Pyruvate Lactate Pyruvate Pyruvate Additions None 10 mM-Lactate 10 mM-Pyruvate

0.31 4.80 1.00

0.024 0.137 5.25

12.9 35.0 0.19

1.37 7.83 3.59

0,087 0.322 4.016

15.7 24.3 0.89

Isolated hepatocytes were prepared from fed animals and incubated as described in the Materials and Methods section. Additions to the incubations were made as shown. After 10 min incubation 0.2 ml samples were applied to silicone oil and cells separated from the medium by centrifugation, as described in the text. Lactate and pyruvate were determined enzymatically. Mean values for two cell preparations are shown.

pyruvate ratios were similar. Incubation of either rat or pig liver cells with I0 mM lactate for 10 min increased the intracellular lactate concentration and significantly increased the lactate/pyruvate ratio. As expected, incubation of either rat or pig liver cells with 10 mM pyruvate for 10 rain increased the intracellular pyruvate concentration and markedly lowered the lactate/pyruvate ratio. Indeed the effect of pyruvate addition on the lactate/pyruvate ratio and hence the calculated cytosolic [NADH]/[NAD +] ratio in pig hepatocytes was greater than in rat hepatocytes. In an earlier report from this laboratory (Clark & Jarrett, 1978) experiments conducted in an attempt to locate the possible site between glucagon-receptor in-

~ 02C

7-

0.I~ t-

0.10

0.05 0.

g

o

~-

o.&~

o.~,

o'.,

',

NADH CONC r" ( m M )

Fig. t. Effects of NADH on membrane low-K, phosphodiesterase activity from pig and rat liver. The membrane fractions containing low-Kin cyclic AMP phosphodiesterase were prepared from pig (O) and rat (0) liver and assayed as described in the Materials and Methods section. The initial substrate concentration was 0.1/~M. Means ___SEM from three animals are given.

teraction and change in glycogenolytic rate, where redox state (in particular pyruvate) exerted its influence, strongly implicated an effect of redox state on cyclic AMP breakdown. As a consequence of that data, properties of the cyclic AMP phosphodiesterases were examined and it was noted that NADH was an inhibitor of the hormone-sensitive membranebound low Km cyclic AMP phosphodiesterase (Clark & Jarrett, 1978). Since exogeneously added pyruvate altered the intracellular pyruvate concentration in pig hepatocytes and lowered the cytosolic [NADH]/[NAD +] ratio, it appeared possible that differences between the species in sensitivity to NADH by the low K~ cyclic AMP phosphodiesterase might exist. Figure 1 shows that the solubilized membrane enzyme (Loten et al., 1978) as isolated from fresh rat liver is significantly less sensitive to NADH than previously shown for the enzyme from isolated rat hepatocytes (Clark & Jarrett, 1978), but does not show significant difference in sensitivity with that prepared from fresh pig liver. The difference in sensitivity to NADH between rat hepatocyte and whole rat liver may reflect impurities in the preparation since the specific activity of the enzyme prepared from isolated rat hepatocytes is approx. 10-fold greater. The failure to detect a difference between the rat and pig enzyme prepared under similar conditions indicates a species similarity but may weaken the general applicability of our previous hypothesis that the regulation of the membrane-bound low Km cyclic AMP phosphodiesterase by the nicotinamide nucleotides provides the molecular basis for the effect of redox state on the hormonal control of hepatocyte metabolism by glucagon. We are currently exploring the possibility that differences in NADH metabolism account for the species differences reported above. Particular attention is being paid to membrane preparations that contain both cyclic AMP phosphodiesterase and NADH dehydrogenase activities.

Acknowledgements---Part of this work was conducted while one of the authors (IGJ) was a Visiting Fellow at the Department of Physiology and Biophysics, Colorado State University, supported by the Kroc Foundation for Medical Research.

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779

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