term (14 h) and short-term (2 h) effects of glucagon on gluconeogenesis and ... glucagon, the periportal cells had a significantly higher rate of ketogenesis (from ...
197
Biochem. J. (1988) 256, 197-204 (Printed in Great Britain)
Glucagon regulation of gluconeogenesis and ketogenesis in periportal and perivenous rat hepatocytes Heterogeneity of hormone action and of the mitochondrial redox state David TOSH, K. George M. M. ALBERTI and Loranne AGIUS* Human Metabolism Research Centre, Department of Medicine, University of Newcastle upon Tyne, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.
Hepatocytes isolated from the periportal or perivenous zones of livers of fed rats were used to study the longterm (14 h) and short-term (2 h) effects of glucagon on gluconeogenesis and ketogenesis. Long-term culture with glucagon (100 nM) resulted in a greater increase (P < 0.01) in gluconeogenesis in periportal than in perivenous cells (93 + 16 versus 30+ 14 nmol/h per mg of protein; 72 % versus 30 % increase), but shortterm incubation (2 h) with glucagon resulted in similar stimulation in the two cell populations. Rates of ketogenesis (acetoacetate and D-3-hydroxybutyrate production) were not significantly higher in periportal cells cultured without glucagon, compared with perivenous cells. However, after long-term culture with glucagon, the periportal cells had a significantly higher rate of ketogenesis (from either palmitate or octanoate as substrate), but a lower 3-hydroxybutyrate/acetoacetate production ratio, suggesting a more oxidized mitochondrial NADH/NAD+ redox state despite the higher rate of fl-oxidation. Periportal hepatocytes had a higher activity of carnitine palmitoyltransferase but a lower activity of citrate synthase than did perivenous cells. These findings suggest that: (i) glucagon elicits greater long-term stimulation of gluconeogenesis in periportal than in perivenous hepatocytes maintained in culture; (ii) after culture with glucagon, the rates of ketogenesis and the mitochondrial redox state differ in periportal and perivenous hepatocytes. INTRODUCTION Parenchymal hepatocytes in the periportal (afferent) and perivenous (efferent) zones of the liver acinus differ in their subcellular structures and enzyme activities. The activities of certain gluconeogenic enzymes are higher in the periportal zone, whereas the activities of some glycolytic and lipogenic enzymes are higher in the perivenous zone (for reviews see [1-3]). This has led to the concept of metabolic zonation, in which different hepatic functions are ascribed to two acinar zones [4]. Although much information has been reported on the heterogeneity of enzyme activities in different zones from studies using histochemical, immunohistochemical and microdissection techniques, it is difficult to extrapolate from differences in activities of enzymes that may not be rate-limiting for a particular pathway to differences in metabolic flux. Recently, techniques have been developed for the isolation of hepatocytes of known specific acinar origin by selective destruction of hepatocytes in either the afferent (periportal) or the efferent (perivenous) zone of the acinus by using digitonin, followed by isolation of hepatocytes from the undestroyed zone [5-7]. This technique enables the measurement of rates of metabolic flux in the isolated periportal and perivenous hepatocytes under defined conditions. It was shown that periportal hepatocytes have higher rates of gluconeogenesis [5,8] and urea synthesis [9], but similar rates of fatty acid synthesis [8], compared with perivenous cells. The differences in rates of gluconeogenesis and urea synthesis persisted when the cells were maintained in monolayer culture [8,9]. *
To whom all correspondence should be addressed.
Vol. 256
To date, hormonal control of liver metabolism has been studied in hepatocytes isolated from the entire liver. It remains to be established whether hepatocytes from different zones of the acinus express the same response to hormones. The purpose of this study was to investigate the regulation of gluconeogenesis and ketogenesis by glucagon in hepatocytes isolated from periportal or perivenous zones of the liver. The results suggest that long-term culture with glucagon causes a greater increase in gluconeogenesis in periportal than in perivenous hepatocytes. The factors that control the heterogeneous expression of the genome in hepatocytes are largely unknown. Hepatocytes isolated from acinar sub-zones and maintained in culture are a potential tool with which to study the induction of metabolic heterogeneity. MATERIALS AND METHODS Materials Digitonin (batch no. 5952180C) was from BDH Chemicals (Poole, Dorset, U.K.). Glucagon and collagenase (Type IV) were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Sources of other chemicals and media were as described previously [10]. Hepatocyte isolation Male rats of the Albino Wistar strain (body wt. 150-200 g) were obtained from the Animal House of the Medical School, University of Newcastle-upon-Tyne, or from Bantin and Kingman (Hull, U.K.). Periportal and perivenous hepatocytes were isolated by the method of
198
D. Tosh, K. G. M. M. Alberti and L. Agius
Quistorff [5], with minor modifications. Digitonin was dissolved (4 mg/ml) in 150 mM-NaCl by warming in a water bath for 15 min. KCl and Hepes were added to final concentrations of 6.7 mm and 50 mm, the pH was adjusted to 7.5 with 2 M-NaOH, and the solution was filtered (0.2 gm-pore-size filter). Sterile perfusion media buffered with Hepes instead of bicarbonate (10 mm for the first two buffers and 30 mm for the collagenase buffer) were used. The duration of digitonin perfusion was determined by the appearance of the destruction pattern [6]. Only livers with uniform destruction patterns were used. After digitonin perfusion, the flow direction of the perfusate was rapidly reversed to prevent digitonin reaching the intact parenchymal cells. The hepatocytes were then isolated by the conventional collagenase perfusion technique [11]. The hepatocytes were dissociated and washed (by centrifugation at 50 g, 2 min) twice in Earle's salts solution and once in medium with 1 % (w/v) defatted bovine serum albumin. Cell yield was (1-2) x 108 cells for periportal preparations and (0.5-1) x 108 for perivenous preparations. Cell viability was > 90 %. In most experiments two periportal and two perivenous preparations were made on each particular day.
Biochemical analysis Acetoacetate, 3-hydroxybutyrate, pyruvate and lactate were assayed by fluorimetric enzymic methods in the deproteinized perchlorate extracts [12]. Palmitate and octanoate were determined by a spectrometric enzymic method [13]. [14C]Glucose was determined in the neutralized perchlorate extract [14]. Cell protein was determined by the Lowry method [15] with bovine serum albumin as standard, and DNA was determined [16] after digestion of RNA with ribonuclease, by using calf thymus DNA as standard. All enzyme activities were determined at 30 'C. Standard methods were used for assay of: alanine aminotransferase (EC 2.6.1.2) [17]; aspartate aminotransferase (EC 2.6.1.1) [17]; lactate dehydrogenase (EC 1.1.1.27) [17]; glutamate dehydrogenase (EC 1.4.1.2) [17]; fructose- 1,6-bisphosphatase (EC 3.1.3.11) [17]; hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) [18]; citrate synthase (EC 4.1.3.7) [19]; carnitine palmitoyltransferase (EC 2.3.1.21) [19]. y-Glutamyltransferase (EC 2.3.2.2) was assayed with a kit (Boehringer Mannheim, 158208). All spectrometric and fluorimetric assays were performed with a Centrifugal Analyser (Cobas Bio, model 8326; Roche, Basle, Switzerland).
Hepatocyte culture Hepatocytes were plated in Minimum Essential Medium containing 5 % (v/v) fetal-bovine serum in 25 cm2 flasks [10]; 4-7 h was allowed for cell attachment for each ofthe four hepatocyte preparations. The medium was then replaced with serum-free Minimum Essential Medium containing 10 nM-dexamethasone [10] either without glucagon or with the concentrations of glucagon described in the text. All test conditions were in duplicate flasks. Two flasks in addition to those used for the metabolic studies were set up for determination of enzyme activities in the hepatocyte monolayers. After cell attachment these monolayers were washed with 150 mM-NaCl and stored at -40 °C for later enzyme
Expression of results Gluconeogenesis (determined from the incorporation of [3-14C]pyruvate into glucose) is expressed as nmol of pyruvate incorporated/h per mg of cell protein. Rates of pyruvate metabolism were determined from the difference in [pyruvate + lactate] at the beginning and end of the 2 h incubation, and are expressed as nmol of pyruvate metabolized/h per mg of cell protein. There was no net accumulation of alanine in the medium, and the rates therefore represent net pyruvate metabolism other than conversion into either lactate or alanine. Rates of palmitate or octanoate uptake were determined from the decrease in [palmitate] or [octanoate] in the medium during a 2 h incubation, and are expressed as nmol of fatty acid taken up/h per mg of cell protein. The rate of formation of ketone bodies (acetoacetate + 3hydroxybutyrate) was determined from the concentration in the medium after a 2 h incubation and is expressed as nmol/h per mg of cell protein. The 3-hydoxybutyrate/ acetoacetate ratio determined after 2 h is also shown. Time-course experiments showed that the rates of substrate utilization and product formation were linear with time during 2 h, and the concentration ratio and thereby the production ratio of 3-hydroxybutyrate/ acetoacetate was constant at 30, 60 and 120 min. Enzyme activities are expressed as munits per mg of protein, where 1 munit is the amount of enzyme converting 1 nmol of substrate/min.
analysis. Metabolic studies The rates of gluconeogenesis (incorporation of [3-14C]pyruvate into glucose) and ketogenesis (formation of acetoacetate and 3-hydroxybutyrate) were determined in monolayers that had been cultured in serum-free medium without or with glucagon for 14 h. In studies on the long-term effects of glucagon, after 14 h culture with the test concentrations of hormone the medium was replaced with fresh Minimum Essential Medium without hormones and supplemented with substrates (palmitate, octanoate, [3-14C]pyruvate or lactate) at the concentrations described in the text. Incubations with these substrates were for 2 h. In studies on the short-term effects of glucagon, hepatocyte monolayers precultured for 14 h were incubated in medium containing substrates either without or with added glucagon for 2 h. On termination of the incubations with substrates, the medium was decanted and a sample deproteinized with HC104 for determination of acetoacetate, 3hydroxybutyrate, pyruvate, lactate and [14C]glucose. Palmitate and octanoate that had not reacted were determined in the rest of the medium. The monolayers were washed and extracted as described previously [121.
Statistical analysis In each of eight experiments, two periportal and two perivenous perparations were made. There was very little intra-experimental variation in the rates of gluconeogenesis or ketogenesis, i.e. between the two periportal or between the two perivenous preparations. The eight experiments were carried out over a period of 4 months, and the inter-experimental variation was significantly higher than the total error (P < 0.05, twoway ANOVA). Some of the data in Tables 2 and 4 include experiments with odd numbers of periportal or
1988
Glucagon action in periportal and perivenous hepatocytes
199
Table 1. Enzyme activities in periportal and perivenous hepatocyte monolayer cultures Hepatocytes were isolated from the periportal or perivenous zone of livers of fed rats and plated as described in the Materials and methods section. Enzyme activities were determined in the monolayers after cell attachment and are expressed as munits per mg of cell protein. Values are means + S.E.M. with the numbers of hepatocyte preparations in parentheses. Statistical analysis was by the Student t test; NS, not significant.
Periportal perivenous
Activity (munits/mg of protein) Periportal hepatocytes Fructose- 1,6-bisphosphatase Alanine aminotransferase Aspartate aminotransferase Lactate dehydrogenase
(23) (23) (23) (23)
y-Glutamyltransferase Glutamate dehydrogenase Citrate synthase Hydroxyacyl-CoA dehydrogenase Carnitine palmitoyltransferase (CPT) CPT (% of glutamate dehydrogenase) CPT (% of citrate synthase)
58.4+2.6 172+8 1551+67 1657+66
(7) 5.1±0.7 (15) 2737+76 (15) 126± 5 (23) 444±14 (15) 24± 1 (15) 0.87±0.05 (15) 19.0+ 1.1
Perivenous hepatocytes (21) (21) (21) (21) (6) (13) (13) (21) (13) (13) (13)
41.0+2.6 117+5 1455 +43 1226 + 90 2.6 +0.7 3117+68 151 +6 459+ 10 19+1 0.61 +0.03 12.8 + 0.8
Ratio
P
1.42 < 0.0005 1.47 < 0.0005 NS 1.35 < 0.0005 1.99 < 0.05 0.87 < 0.005 0.83 < 0.005 NS 1.26 < 0.005 1.43 < 0.0005 1.48 < 0.0005
Table 2. Rates of gluconeogenesis in periportal and perivenous hepatocyte cultures incubated with different substrates
Periportal and perivenous hepatocytes were pre-cultured for 14 h either without or with 100 nM-glucagon and then incubated for 2 h with fresh medium without glucagon, containing the substrates indicated. The concentrations of pyruvate, palmitate and L-carnitine were respectively 0.7 mm, 0.75 mm and 0.5 mm. Rates of gluconeogenesis from [3-'4C]pyruvate are expressed as nmol of pyruvate incorporated into glucose/h per mg of cell protein, and are means + S.E.M. for the numbers of hepatocyte preparations shown in parentheses. Values for ' + glucagon' cultures that are significantly different from the corresponding controls (paired t test) are shown: **P < 0.0001.
Periportal
Hepatocytes...
Substrates
14 h pre-culture ...
Pyruvate Pyruvate + palmitate Pyruvate + palmitate + carnitine
Control
RESULTS Enzyme activities, protein and DNA Enzyme activities were determined in periportal and perivenous hepatocytes after cell attachment in culture. In agreement with previous studies using the digitonin/ collagenase perfusion technique [5,8,9], hepatocytes from the periportal zone had a higher activity of alanine aminotransferase, lactate dehydrogenase and yglutamyltransferase and a lower activity of glutamate
Control
38+ 14 (5) 9+ 3 70±35 (4) 20+ 11 241 +27** (14) 100+ 13
(6) 15+5 (4) 42+19 (14) 140+ 18
perivenous preparations. All other data (Fig. 1 and Table 2) and studies with either octanoate or palmitate and pyruvate comprise only experiments with two periportal and two perivenous preparations each. Differences between periportal and perivenous data from sets comprising a minimum of six experiments (12 periportal and 12 perivenous) were analysed by two-way analysis of variance. Other experiments (n < 12) were analysed by the unpaired Student t test. Differences between rates in the presence of glucagon and the respective controls were analysed by the Student paired t test.
Vol. 256
+ Glucagon
Perivenous + Glucagon
16+4 39 +24 130+ 17
dehydrogenase than did perivenous hepatocytes (Table 1). The activity of fructose-1,6-bisphosphatase was higher in periportal cells, but the activity of hydroxyacylCoA dehydrogenase was similar in the two cell populations (Table 1). The distribution of citrate synthase and carnitine palmitoyltransferase in the periportal and perivenous zones of the liver has not been reported previously. Citrate synthase activity was higher in perivenous cells, and carnitine palmitoyltransferase activity was higher in periportal cells (Table 1). The cell protein/DNA ratio determined after 14 h of culture was similar in periportal and perivenous hepatocytes (periportal, 72.4+ 7.8, n = 10; perivenous, 73.0+ 5.5, n 10, means + S.E.M.). Throughout this study metabolic rates are expressed in relation to cell protein. Long-term effects of glucagon on gluconeogenesis We compared the rates of gluconeogenesis in periportal and perivenous hepatocytes cultured for 14 h either without or with glucagon, from the incorporation of [3-14C]pyruvate into glucose in the presence of 5 mMglucose. The rate of pyruvate metabolism was also =
200
D. Tosh, K. G. M. M. Alberti and L. Agius
determined enzymically. In preliminary studies, gluconeogenic rates were determined with different substrates. Rates were low when determined in the presence of pyruvate alone, but higher rates were obtained in the presence of palmitate and carnitine, but
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Fig. 1. Dose-response curve to glucagon of gluconeogenesis in periportal and perivenous hepatocytes Hepatocytes isolated from the periportal or perivenous zones of rat liver were cultured with the concentrations of glucagon shown for 14 h. Gluconeogenesis was determined during a subsequent 2 h incubation in hormone-free medium containing 0.6 mM-[3-14C]-pyruvate, 0.75 mMpalmitate and 0.5 mM-L-carnitine. Rates of gluconeogenesis are expressed as nmol of pyruvate incorporated into glucose/h per mg of cell protein. Values are means and S.E.M. bars for 14 periportal (PP, *) and 14 perivenous (PV, A) preparations. Significant differences between periportal and perivenous values are shown by: *P < 0.01; **P < 0.005.
not in the presence of palmitate alone (Table 2). In the latter case the low endogenous concentration of carnitine limits mitochondrial palmitate metabolism [10]. In the rest of this study, rates of gluconeogenesis from pyruvate were determined in the presence of 0.75 mM-palmitate and 0.5 mM-L-carnitine. The effects of 14 h culture with 1-100 nM-glucagon on the rates of gluconeogenesis are shown in Fig. 1, and the incremental effects of the hormone are shown in Table 3. Glucagon caused a doserelated stimulation of gluconeogenesis of 720 in periportal and 30 0 in perivenous cells. Gluconeogenic rates were significantly higher in periportal than in perivenous cells when pre-cultured either without or with glucagon (1-100 nM). The incremental increase by glucagon was significantly higher (2-3-fold) in periportal than in perivenous cells (Table 3). In the experiments shown in Fig. 1, glucagon also increased total pyruvate metabolism, assessed from the decline in pyruvate and lactate concentration in the medium in both periportal (control 418 + 38, 1 nmglucagon 473+36, 10 nM-glucagon 529+65, 10 nMglucagon 541 + 44 nmol of pyruvate/h per mg of protein; means+ S.E.M., n = 12) and perivenous cells (control 350 + 25, 1 nM-glucagon 379 + 27, 10 nM-glucagon 418+ 58, 100 nM-glucagon 428 + 53; means+S.E.M., n = 12). Values for periportal cells cultured for 14 h with 100 nM-glucagon were significantly higher than corresponding values for perivenous cells (P < 0.025). The rates of gluconeogenesis (determined radiochemically) were 41 + 4 % and 50 + 5 % of the rates of total pyruvate metabolism (determined enzymically) for periportal cells precultured without or with 100 nM-glucagon respectively, and 35 + 2 % and 35 + 500 for perivenous cells cultured without or with glucagon. Values for periportal cells cultured with glucagon were higher (P < 0.03) than values for perivenous cells. Short-term effects of glucagon on gluconeogenesis The short-term effects of glucagon on gluconeogenesis were studied in hepatocytes pre-cultured for 14 h without glucagon and then incubated for 2 h with pyruvate, palmitate and carnitine either without or with 100 nM-
Table 3. Effects of glucagon on gluconeogenesis and ketogenesis after 14 h or 2 h culture in periportal and perivenous hepatocytes For experimental details see the legend to Fig. 1. Rates of gluconeogenesis in the control cultures and the changes in rates caused by glucagon are expressed as nmol of pyruvate incorporated/h per mg of protein. Rates of ketogenesis and the changes caused by glucagon are expressed as nmol of acetoacetate + 3-hydroxybutyrate formed/h per mg of protein. The ratios of the production rate§ of 3-hydroxybutyrate and acetoacetate are shown; minus signs indicate a decrease relative to controls. Values are means + S.E.M. for 14 periportal and 14 perivenous preparations. Effects of glucagon that are significantly different (unpaired t test) between periportal and perivenous preparations are indicated by: *P < 0.05; **P < 0.01.
Gluconeogenesis
Hepatocytes... Rates of controls
Ketogenesis Perivenous Periportal
Periportal
Perivenous
140+18
100+ 13
385 + 35
14+0.3 43+9 93+ 16 132+26
6+7 15+5*
39+12 90+24 152+ 23 117+44
3-Hydroxybutyrate/ acetoacetate
Periportal
Perivenous
340+ 32
2.74+0.31
2.86+0.26
25 + 7 58+17 90 + 16* 136+ 28
-0.43 +0.14 - 1.10+0.21 -1.53 +0.23 -0.95 +0.22
0.00 +0.10* -0.54+0.16* -1.12+0.17 -0.75 +0.14
Change caused by:
14 h culture with: I1 nM-glucagon 10 nM-glucagon 100 nM-glucagon 2 h culture with 100 nm-
30+ 14** 77+ 14
glucagon
1988
Glucagon action in periportal and perivenous hepatocytes
201
Table 4. Rates of ketogenesis in periportal and perivenous hepatocytes incubated with different substrates
Periportal and perivenous hepatocytes were pre-cultured for 14 h either without or with 100 nM-glucagon and then incubated for 2 h with the fresh medium without glucagon containing the substrates indicated below. The concentrations of lactate and pyruvate were 0.6 mm, palmitate 0.75 mm, and carnitine 0.5 mm. Rates of ketogenesis are expressed as nmol of acetoacetate + 3hydroxybutyrate formed/h per mg of protein. The ratio of the production rate of 3-hydroxybutyrate/acetoacetate is also shown. Values are means+S.E.M. The number of periportal (PP) and perivenous (PV) preparations for each substrate is shown in parentheses. Values for cells precultured with glucagon that are significantly different from the respective controls (t test) are shown by: *P < 0.05; **P < 0.005. Ratio of 3-hydroxybutyrate/acetoacetate production
Production of acetoacetate + 3-hydroxybutyrate (nmol/h per mg of protein)
Hepatocytes ...
Perivenous
Periportal
14 h pre-culture... Control + glucagon Substrates (n: PP,PV)
Control + glucagon
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Control
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0.64+0.13 0.33+0.07* 0.59+0.07 0.39+0.03* 1.07+0.15 0.41 +0.07* 0.87+0.14 0.44+0.03 2.74+0.31 1.21 + 0.11 ** 2.86+0.26 1.78 + 0.25**
62+11* 42+5 32+2 54+8 Pyruvate (6,5) 53+ 11 55+ 14 Pyruvate+palmitate (4,3) 66+ 15 62+ 15 385 + 35 537+45** 340+ 32 443 +46** Pyruvate + palmitate + carnitine (14,14) 473+52 617+52** 387+32 490+53* Lactate+palmitate+ carnitine (6,6) 575+55 762+66** 498+54 599+63** Octanoate (12,12)
_ 600-
Perivenous
Periportal
3.84+0.38
2.04+0.16*
3.28+0.49
1.63+0.19*
2.32+0.18
1.78+0.18** 2.44+0.20 2.04+0.16**
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Fig. 2. Dose-response curve to glucagon of ketogenesis (a) and the production ratios of 3-hydroxybutyrate/acetoacetate (b) in periportal and perivenous hepatocytes Hepatocytes isolated from the periportal or perivenous zones of rat liver were cultured with the concentrations of glucagon shown for 14 h. Rates of ketogenesis were determined during a subsequent 2 h incubation in hormone-free medium containing 0.5 mM-palmitate, 0.5 mM-carnitine and 0.6 mM-pyruvate. Rates of ketogenesis are expressed as nmol of acetoacetate +3hydroxybutyrate formed/h per mg of protein (a) or as the ratio of the production rates of 3-hydroxybutyrate/acetoacetate (b). Values are means and S.E.M. bars for 14 periportal (PP, *) and 14 perivenous (PV, A) preparations. Significant differences between periportal and perivenous values are shown by: *P < 0.05; **P < 0.01.
glucagon. Glucagon stimulated gluconeogenesis by 82 % in periportal cells [control 172+ 17, 100 nM-glucagon (2 h) 313+37 nmol of pyruvate/h per mg of protein; means+ S.E.M., n = 12] and by 70 % in perivenous cells [control 127+10, l0OnM-glucagon (2h) 216+15; means+S.E.M., n= 12]. The incremental increase by incubation with glucagon for 2 h was not significantly different between the two cell populations (Table 3), although the rates were still significantly higher (P < 0.05, unpaired t test) in the periportal cells than in the perivenous. Long-term effects of glucagon on ketogenesis The rates of ketogenesis in periportal and perivenous hepatocytes precultured for 14 h without or with 100 nMglucagon and then incubated for 2 h with substrates Vol. 256
(without glucagon) are shown in Table 4. With pyruvate the only exogenous substrate, or with pyruvate and palmitate (without carnitine), the rates of ketogenesis were low compared with the rates either with palmitate + carnitine or with octanoate (Table 4). In cells pre-cultured with 100 nM-glucagon (14 h), rates of ketogenesis (from either palmitate and carnitine or octanoate) were higher than the respective controls cultured without glucagon, for both periportal and perivenous preparations (Table 3), indicating that longterm culture with glucagon increases ketogenesis from as
these substrates. The effects of 14 h culture with 1100 nM-glucagon on the rates of ketogenesis determined during a subsequent 2 h incubation are shown in Fig. 2, and the incremental effects of the hormone are shown in Table 3. Glucagon caused a dose-related stimulation of
202
ketogenesis of 40 % and 30 % in periportal and perivenous cells respectively (Fig. 2, Table 3). The rates of ketogenesis in periportal cells cultured without glucagon were slightly, but not significantly, higher than in perivenous cells. However, in cells cultured with glucagon (1-100 nM), rates of ketogenesis from either palmitate and carnitine (Fig. 2) or octanoate (Table 4) were significantly higher in periportal than in perivenous cells (P < 0.01 for incubations with palmitate and carnitine and P < 0.025 for incubations with octanoate). Short-term effects of glucagon on ketogenesis The short-term stimulation of ketogenesis by glucagon (100 nM) during a 2 h incubation with palmitate (0.75 mM), carnitine (0.5 mM) and pyruvate (0.6 mM) in cells precultured for 14 h without the hormone was 23 % in periportal cells [control 441 + 42, 100 nM-glucagon (2 h) 544 + 62 nmol of ketone bodies/h per mg of protein; means+ S.E.M., n = 9] and 31 % in perivenous cells [control 406+29, 100 nM-glucagon (2 h) 530+51; means + S.E.M., n = 9]. Values for periportal cells were not significantly different from the corresponding values for perivenous cells. Palmitate and octanoate uptake The rates of palmitate uptake (determined from the rate of decrease in [palmitate] in the medium) were increased by 14 h culture with 100 nM-glucagon (P < 0.005, paired t test) in both periportal (control 193+ 19, 100 nM-glucagon 257 + 23 nmol of palmitate/h per mg of protein; means+ S.E.M., n = 12) and perivenous cells (control 150+19, 100nM-glucagon 187+24). In cells cultured with glucagon, but not in controls, the rates were significantly higher in periportal than in perivenous cells (P < 0.05). The rates of octanoate uptake showed similar trends (periportal: control 303 + 42; 100 nMglucagon, 400 + 43; perivenous: control, 237 + 34; 100 nM-glucagon, 285 + 38; means + S.E.M., n = 12; nmol of octanoate/h per mg of protein) and were higher after culture with glucagon in periportal than in perivenous cells (P < 0.01). Long-term effects of glucagon on 3-hydroxybutyrate/ acetoacetate ratios The ratio of 3-hydroxybutyrate/acetoacetate concentrations in the medium after 2 h, which reflects the production rates of these two ketone bodies (see the Materials and methods section), was low with substrates which gave low rates of ketogenesis (pyruvate alone or pyruvate and palmitate) compared with incubations with palmitate and carnitine or octanoate (Table 4). For all substrates, the 3-hydroxybutyrate/acetoacetate ratios were decreased by 14 h pre-culture with glucagon. Fig. 2(b) shows the effects of [glucagon] during the 14 h preculture on the 3-hydroxybutyrate/acetoacetate ratio in periportal and perivenous hepatocytes. Although there was no significant difference in the ratios between periportal and perivenous preparations cultured without glucagon, the ratios were significantly lower in the periportal cells after culture with 1-100 nM-glucagon (Fig. 2b). Short-term effects of glucagon on 3-hydroxybutyrate/ acetoacetate ratios When hepatocyte monolayers were precultured without glucagon (14 h) and then incubated for 2 h with
D. Tosh, K. G. M. M. Alberti and L. Agius
palmitate, pyruvate and carnitine, either without or with 100 nM-glucagon, the production ratio of 3-hydroxybutyrate/acetoacetate was significantly decreased by glucagon (P < 0.005, paired t test) for both periportal [control 2.50+0.26, 100 nM-glucagon (2 h) 1.51+0.09; means+ S.E.M. n = 9] and perivenous hepatocytes (control 2.73 +0.18, 100 nM-glucagon (2 h) 1.87 +0.13; means+S.E.M._ n = 9]. Values for periportal cells incubated with glucagon were lower (P < 0.04, t test) than the corresponding ratios of perivenous cells. When the short-term effects of glucagon (2 h) were examined in hepatocyte monolayers pre-cultured for 14 h with 100 nM-glucagon, there was a small but significant further decrease in the 3-hydroxybutyrate/acetoacetate production ratio [periportal cells: control, 1.10 + 0.10, glucagon (2 h), 0.95 + 0.05; perivenous: control, 1.51 + 0.13; glucagon (2 h), 1. 17 + 0.06; means + S.E.M., n = 9, 9 respectively]. Also in these experiments, values for periportal cells were significantly lower than perivenousvalues[control,P < 0.03;glucagon(2 h),P < 0.02; t test]. Effects of substrate concentration
The decrease in the 3-hydroxybutyrate/acetoacetate production ratio in cells cultured with glucagon may be due either to a more oxidized mitochondrial NADH/NAD+ redox state, if the 3-hydroxybutyrate dehydrogenase reaction is near equilibrium [20], or to the increase in production rate of acetoacetate if the activity of the dehydrogenase is insufficient to maintain equilibrium. To distinguish between these possibilities, hepatocytes precultured either without or with glucagon (100 nM) were incubated with a range of palmitate concentrations (0.25-1.0 mM) to achieve a range of rates of ketogenesis. In these experiments glucagon decreased the 3-hydroxybutyrate/acetoacetate production ratio at all palmitate concentrations examined; however, it increased ketogenesis only at the higher concentrations (0.5-1 mM) (D. Tosh & L. Agius, unpublished work). This indicates that the decrease in the 3-hydroxybutyrate/ acetoacetate ratio was independent of the production rate of acetoacetate.
DISCUSSION Heterogeneity of enzyme activities In the present study a similar heterogeneity of glutamate dehydrogenase, alanine aminotransferase and lactate dehydrogenase in periportal and perivenous hepatocytes was observed as in previous studies using the digitonin/collagenase perfusion technique [5,8,9,21]. The heterogeneity of fructose- 1,6-bisphosphatase observed in the present study agrees with data from micro-dissection studies [22]. The distribution of citrate synthase and carnitine palmitoyltransferase in different zones of the liver acinus has not been reported previously; the activity of citrate synthase was higher in perivenous cells, but that of carnitine palmitoyltransferase activity was higher in periportal cells. Citrate synthase is involved in the entry of acetyl-CoA into the citrate cycle and in the transfer of mitochondrial acetyl-CoA to the cytoplasm for lipogenesis. Micro-dissection studies have shown that succinate dehydrogenase is higher in the periportal zone [23], suggesting a higher capacity for the citrate cycle in this zone [2,3]. The perivenous zone has higher activities of acetyl-CoA carboxylase [24] and ATP citrate lyase 1988
Glucagon action in periportal and perivenous hepatocytes
[25], suggesting a higher capacity for lipogenesis. The higher activity of citrate synthase in perivenous hepatocytes may be associated with a higher rate of mitochondrial formation of citrate for lipogenesis rather than entry of acetyl-CoA into the citrate cycle. The higher activity of carnitine palmitoyltransferase in periportal cells suggests a higher capacity for the mitochondrial metabolism of fatty acids, and may in part explain the higher rates of ketogenesis in periportal hepatocytes cultured with glucagon. Glucagon action in periportal and perivenous hepatocytes The higher rate of gluconeogenesis in periportal than in perivenous hepatocytes agrees with the findings of Quistorff [5,8] on hepatocytes from fasted rats and shows that the heterogeneity of gluconeogenesis is also present in the fed state. The possibility that hepatocytes isolated from the periportal and perivenous zones from rat liver may express different responses to hormones has not been investigated previously. This study shows that glucagon causes greater long-term stimulation of gluconeogenesis and ketogenesis in periportal than in perivenous cells. This may be due to differences in glucagon receptor number or to differences in post-receptor events. The former possibility seems unlikely, since the short-term effects of glucagon on gluconeogenesis and ketogenesis were similar in the two cell populations. Since the periportal zone of the liver has a higher density of rough endoplasmic reticulum than the perivenous zone [11, long-term changes in enzyme induction may be more prominent in periportal cells. It has been proposed that metabolic heterogeneity in vivo may result from concentration gradients of substrates, oxygen and hormones across the portal/venous vasculature [2,3]. The present findings raise the question whether intrinsic differences in long-term responses to hormones may also contribute to heterogeneity of enzyme expression in hepatocytes in different zones. Mitochondrial fatty acid metabolism Quistorff and co-workers found no difference in rates of fatty acid synthesis between periportaI and perivenous hepatocytes isolated from fasted rats [8]. In the present study rates of ketogenesis from either palmitate or octanoate were slightly higher in periportal hepatocytes, but the difference was smaller than the difference in gluconeogenesis and was statistically significant only after culture with glucagon. The higher rate of ketogenesis in cells cultured with glucagon was not due to an increased ATP requirement for gluconeogenesis, since it was also observed when the hepatocytes were incubated without gluconeogenic substrates. It may be related to the higher activity of carnitine palmitoyltransferase, or to intramitochondrial differences distal to carnitine palmitoyltransferase, since ketogenesis from octanoate (which can enter mitochondria independently of carnitine palmitoyltransferase) was also higher in periportal cells. 3-Hydroxybutyrate/acetoacetate ratios and the mitochondrial redox state The 3-hydroxybutyrate/acetoacetate production ratios in hepatocyte monolayers incubated with palmitate and carnitine were in the same range as the values reported for freeze-clamped liver t20]. The, stimulation of ketogenesis by glucagon after either long-term (14 h) Vol. 256
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culture or short-term (2 h) incubations was associated with an increased production rate of acetoacetate and a decreased production rate of 3-hydroxybutyrate. This effect of glucagon was independent of the rate of ketogenesis (determined as a function of palmitate concentration), suggesting that it was not due to a limiting activity of 3-hydroxybutyrate dehydrogenase at high turnover rates of acetoacetate and probably reflects the mitochondrial redox state. Glucagon lowered the 3hydroxybutyrate/acetoacetate ratio more markedly in the presence of a gluconeogenic substrate (irrespective of whether this was pyruvate or lactate), although in the absence of these substrates a significant decrease was also observed. The stimulation of gluconeogenesis by glucagon may contribute to the more oxidized mitochondrial redox state, either through an increased ATP requirement for gluconeogenesis or by increased transfer of reducing equivalents from the mitochondrial to the cytoplasmic compartment for gluconeogenesis. Glucagon may induce a more oxidized mitochondrial NADH/NAD+ redox state by stimulating electron transport [26]. A similar effect of glucagon on the 3hydroxybutyrate/acetoacetate ratio was shown by Christiansen [27] in freshly isolated hepatocyte suspensions incubated in Ca2l-free medium, but not in the presence of 1 mM-Ca2". Possible explanations for these observations are that freshly isolated hepatocytes may be more permeable to Ca2" than the cultured cells, or that glucagon may increase the permeability to Ca2" in freshly isolated, but not in cultured, hepatocytes. Heterogeneity of the mitochondrial redox state The simplest explanation for the observation that there was no significant difference in the 3hydroxybutyrate/acetoacetate ratio between periportal and perivenous hepatocytes cultured without glucagon, but the ratio was significantly lower in periportal cells after culture with glucagon, is that glucagon may cause a greater stimulation of electron transport in periportal than in perivenous cells. Histochemical studies have suggested that cytochrome oxidase activity may be higher in the periportal zone [28]. Glucagon may induce a greater stimulation of electron transport in periportal cells if these cells have a higher cytochrome activity. It is generally assumed in studies on the 3-hydroxybutyrate/acetoacetate ratio in freeze-clamped liver that the mitochondrial NADH/NAD+ ratio is uniform throughout the liver lobule. In studies where the rates of ketogenesis in periportal and perivenous zones of the liver lobule have been determined from changes in surface fluorescence in the intact perfused liver [29], it has also been assumed that the mitochondrial redox state is homogeneous throughout the liver lobule [30,31]. However, there is evidence for a more oxidized NADH/ NAD+ redox state in the periportal zone of the perfused liver, from fluorescence scanning of oxidized flavoprotein/NADH [32]. The present finding of a lower hydroxybutyrate/acetoacetate ratio in periportal cells cultured with glucagon (despite their higher rate of ketogenesis) supports the observations in perfused liver [32]. Heterogeneity of the mitochondrial redox state has important implications in the interpretation of changes in surface flourescence [30,31]. Rates of ketogenesis in periportal and perivenous zones of the intact perfused liver have been estimated by assuming that surface NADH fluorescence is directly proportional to
204
ketogenesis, and that differences in basal flourescence in different zones reflect differences in quenching and not differences in redox state. Because of the latter assumption, changes in rates of ketogenesis have been estimated from percentage, as opposed to absolute, changes in fluorescence [30,31]. The present study shows that in periportal and perivenous hepatocytes the redox state does not parallel the rate of ketogenesis. It is therefore more valid to study the regulation of the redox state on hepatocyte sub-populations than on the conventional heterogeneous population isolated from whole liver. This work was supported by Grants from the University of Newcastle Research Committee, the British Diabetic Association and the Peel Medical Research Trust. D. T. holds a M.R.C. studentship. We thank Dr. P. D. Home and Dr. H. A. W. Neil for advice on statistical analysis.
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Received 11 March 1988/20 June 1988; accepted 6 July 1988
1988
