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Propionate metabolism and its regulation by fatty acids in ovine hepatocytes
Comp. Biochem. Physiol. Vol. 84B, No. 4, pp. 559-563, 1986 Printed in Great Britain
0305-0491/86 $3.00+ 0.00 PergamonJournals Ltd
P R O P I O N A T E M E T A B O L I S M A N D ITS R E G U L A T I O N BY F A T T Y ACIDS IN O V I N E H E P A T O C Y T E S ANNE FAULKNER* and HELEN T. POLLOCK Hannah Research Institute, Ayr KA6 5HL, Scotland (Tel: 0292-76013) (Received 7 November 1985)
Abstract--1. Propionate metabolism was studied in ovine hepatocytes. 2. The main products of metabolism were CO2, glucose, L-lactate and pyruvate. 3. The fatty acids, butyrate and palmitate inhibited propionate oxidation; butyrate inhibited but palmitate slightly stimulated gluconeogenesis from propionate. 4. Butyrate and palmitate also inhibited lactate and pyruvate production from both endogenous substrates and from propionate.
Propionate is produced by fermentation in the rumen and is a major precursor for glucose synthesis in the ruminant liver (Bergman, 1973). Over 90% of the propionate transported via the portal vein is extracted by the liver and it has been estimated that glucose synthesis from propionate could account for 50-60% of the glucose produced in the ruminant (Leng, 1970; Bergman, 1973). The pathway for glucose synthesis from propionate is well-established but little is known of the control mechanisms which regulate this pathway in the ruminant liver. As the ruminant absorbs little glucose from the diet, relying almost exclusively on gluconeogenesis for its glucose supply, rates of glucose synthesis are highest in the fed animal, decreasing during fasting (Bergman, 1973). This contrasts with the non-ruminant, where fasting stimulates gluconeogenesis. Hence the factors regulating gluconeogenesis in ruminant and non-ruminant species may be expected to differ markedly. The present paper describes studies on propionate metabolism in ovine hepatocytes and its regulation by the fatty acids, butyrate and palmitate, both of which are taken up by the sheep liver in vivo (Bergman and Wolff, 1971; Katz and Bergman, 1969).
Amersham International, Aylesbury, Bucks. Chemicals were from BDH Chemicals, Dagenham, Essex. Methods Hepatocytes were prepared by perfusing the caudate lobe with a collagenase solution (0.5% w/v) as previously described (Clark et al., 1976). Hepatocytes (~5mg dry wt) were incubated in a final volume of 2.5 ml of Krebs Ringer bicarbonate buffer gassed with 95% 02 and 5% CO2 containing 1.5% (w/v) gelatin and 1% (w/v) fatty-acid free albumin and incubated for 2 hr with shaking at 37°C. Reactions were stopped by the addition of HC1 04. Glucose, lactate, pyruvate, citrate, 2-oxoglutarate and malate were determined by enzymatic analysis (see Bergmeyer, 1973). Radioactive glucose was isolated and determined as the pentacetate (Jones, 1965). Production of ~4CO2 was determined by trapping CO2 in hyamine hydroxide following acidification of the incubation medium (Whitelaw and Williamson, 1977). Radioactivity at 0 min was determined routinely and subtracted from experimental values. Propionate was determined by glc. Statistical methods Results are expressed as means+SEM. Statistical analysis was performed using Student's t-test for paired observations.
RESULTS MATERIALS AND METHODS Animals Sheep were Finn x Dorset Horn cross-breeds. They were 1-4 years old and were fed hay ad lib plus a cereal mix (300400 g/day depending on age). A catheter was placed in the jugular vein on the day before the experiment, the sheep were then anaesthetized with an intravenous injection of sodium pentabarbitone before the caudate lobe of the liver was removed. Animals were killed before they recovered from anaesthesia. Materials Enzymes and biochemicals were obtained from Boehringer Corp., Lewes, Sussex and Sigma Chemical Co., Poole, Dorset. Radiochemicals were obtained from
*Address for correspondence: Department of Lactational Physiology and Biochemistry, Hannah Research Institute, Ayr, Scotland.
Metabolism o f propionate in ovine hepatocytes
Substantial quantities of [1-J4C]propionate were taken up by ovine hepatocytes and, following incubation, most of the radioactivity from the metabolized propionate was recovered in glucose and CO 2 (Table 1). In a second experiment, radioactive glucose production was determined using 14[C]NaHCO3 and [2-14C]propionate as well as [1-14C]propionate. Rates of radioactive glucose formation from [14C]NaHCO3 were similar to those from [1-~4C]propionate, but those from [2-~4C]propionate were more than double (Table 2). This is to be predicted as radioactivity from [l-14C]propionate and [14C]NaHCO3 equilibrate on the formation of succinate, and half of this radioactivity is lost subsequently at the phosphoenol pyruvate carboxykinase reaction. The radioactive CO2 lost at this step (equivalent to that incorporated into glucose for [1-14C]propionate) was subtracted from
559
560
ANNE FAULKNER and HELEN T, POLLOCK Table 1. [I-~4C]Propionate metabolism and its regulation b v bukvratc and palmitate , mo]
[I-
Propionate
.
.
.
.
.
~'2] prc~pi~!n:l~o/K ,]rv Wt , nf.ll ''~r
+ ~llt,,,r, a t e
+ palmit=+,,
.?t~
Conversion ef [!-t"C] pPopionate to:
l~ro
494
[~C:]
g]ucose
~ ~4
'?~,
6/)
I~
:£,*
11~
I~m
Hepatocytes were incubated as described in Materials and Methods section. Propionate was present at a concentration of 3 raM, butyrate at 3 mM and palmitate at 1 raM. Incubations containing palmitate also contained I mM carnitine. Results significantly differen t from propionate alone are expressed by *P <0.05. **P < 0.01, ***P < 0.001, n - 12.
the total ~4CO2 production to give values for the net production of CO 2 from [l-14C]propionate (Table 2).
proportion metabolized to [~4C]glucose (14% control. 15% butyrate, 22% palmitate).
Effects of Jree fatty acids on propionate metabolism The fatty acids butyratc and palmitate reduced significantly the uptake of propionate by the hepatocytes (Table 1), and the total and net J4CO2 production from [1-14C]propionate (Tables 1 and 2). Production of J4CO2 from [2-14C]propionate was also decreased slightly but not significantly (Table 2). Incorporation of radioactivity into glucose from all three precursors was significantly reduced by butyrate (Tables 1 and 2); palmitate significantly increased incorporation (Tables 1 and 2). The proportion of the [1-~4C]propionate taken up by the hepatocytes which was metabolized to ~4CO2 and glucose was also affected by free fatty acids. The proportion oxidized to 14CO2 decreased from 75% in the control to 64 and 62% in the presence of butyrate and palmitate respectively; palmitate but not butyrate increased the
Lactate and pyruuate production in ovine hepatocytes Lactate and pyruvatc were produced in ovine hepatocytes incubated without exogenous substrate (Table 3), presumably originating from breakdown and glycolysis of glycogen. The addition of propionate increased the amount of lactate and pyruvate produced (Table 3) and decreased the ratio of lactate to pyruvate (Table 4). The presence of butyrate or palmitate significantly reduced the amount of lactate and pyruvate produced by the hepatocytes both from endogenous substrate and the net amount produced from propionate (Table 3). The fatty acids had no significant effect on the ratio of lactate to pyruvate in the absence of propionate but increased the ratio in the presence of propionate (Table 4). The production of glucose, CO2, lactate and pyruvate accounted for 103, 87 and 92% of the propionate
Table 2. Glucose and (:02 production from radioactive precursors Hmol
suhstrate
Proplona~e
ineorporateJ/g
dr}' wt, cell/>hr
÷ butvrate
+ pilmit
~te
alone [I"C]
glucose
from:
[I-I"C] peopionate
l~a
[2-i"C]
~6% t 80
proptonate
[I.C]NaHCO
18%
' 2?
, %%
l!6
!q**i
15<
!~3F 12 c:
16"*
%8**
I):
t"C02 from: [1-t~'C] propionate
%QQ " 8~
27b
[2-I"C] propionate
38 + 13
~2
Net
i"CO
[I-I"c]
6?* 10
from:
pPopionate
246
~ 65
1 ~1
35*
Hepatocytes were incubated as described in Materials and Methods section. Propionate was present at a concentration of 5 raM, butyrate at 3 mM and palmitate at 1 mM. Incubations containing palmitate also contained l m M carnitine. Nel ~4CO2 production from [IJ4C]propionate was calculated as the difference between total ~4CO2 production and ~4C incorporation into glucose. Results significantly different from propionate alone are expressed by *P <0.05, **P <0.01, ***P < 0.001, n -= 7.
Propionate metabolism in ovine hepatocytes
561
Table 3. Effect of butyrate and palmitatc on lactate and pyruvate production Lactate plus pyruvate production (~mol/g dry wt. cells/ 2hr)
÷ butyrate
Endogenous production
Net production from propionate
÷ palmltate
19~ ± 25
47 ± 7"*
79 ± 27*
86 ± 35
33 ± 11"
~3 ± 16"
Hepatocytes were incubated as described in the Materials and Methods section. Propionate was present at 3mM, butyrate at 3mM and palmitate at l mM. Carnitine (l mM) was included in incubations containing palmitate. Endogenous production is that produced in the absence of propionate. Net production from propionate was calculated as the difference between total lactate and pyruvate production in the presence and absence of propionate. Results significantlydifferent from rates in the absence of butyrate or palmitate are expressed by *P < 0.05, **P < 0.01, n = 12. t a k e n u p by the hepatocytes in the absence o f free fatty acid, the presence o f b u t y r a t e or palmitat¢ respectively (Tables 1 a n d 3).
determine w h e t h e r the effects o f free fatty acids o n lactate a n d pyruvat¢ p r o d u c t i o n were due to decreased rates of p r o d u c t i o n or increased rates o f utilization. As m i g h t be predicted a - c y a n o h y d r o x y cinnamic acid significantly ( P < 0.01) increased the a m o u n t o f lactate a n d p y r u v a t e p r o d u c e d by ovine hepatocytes in all i n c u b a t i o n s (Table 6), indicating t h a t some lactate a n d pyruvat¢ p r o d u c e d was further metabolized normally. Butyrate a n d palmitate reduced lactate a n d p y r u v a t e p r o d u c t i o n f r o m e n d o g e n o u s substrate a n d a l m o s t eliminated the net p r o d u c t i o n f r o m p r o p i o n a t ¢ in b o t h the presence a n d absence o f the inhibitor (Table 6). This indicates t h a t there is a n effect o f these free fatty acids o n lactate a n d p y r u v a t e production. In addition, if the difference in the rate o f lactate a n d p y r u v a t e
Carboxylic acids in ovine hepatocytes after incubation W h e n hepatocytes were i n c u b a t e d with p r o p i o n a t e a n d either of the free fatty acids, the c o n c e n t r a t i o n s o f the carboxylic acids in the cells increased substantially (Table 5). T h e fatty acids alone did n o t produce this effect; p r o p i o n a t e p r o d u c e d increases in intracellular m a l a t e only (Table 5).
Effect of ct-cyanohydroxycinnamic acid on lactate and pyruvate production This i n h i b i t o r o f p y r u v a t e t r a n s p o r t to the mitoc h o n d r i a (Halestrap a n d D e n t o n , 1974) was used to
Table 4. Effect of butyrate and palmitate on the ratio oflactate to pyruvate produced by ovine hepatocytes Lactate
÷ butyrate
So addition
No substrate
Propionate
: pyruvate
+ palmitate
13.1
12.8
14.9
4.4
8.1
9.5
Hepatocytes were incubated as described in the Materials and Methods section. Propionate was present at 3 mM, bntyrate at 3 mM and palmitate at ! mM. Carnitine (1 mM) was included in incubations containing palmitate. Table 5. Concentrations of carboxylic acids in ovine hepatocytes Cohen.
(nmol/g dry wt. hepatoeytes)
2-oxoglutarate
malate
citrate
Proplonate
342 ± 89
861 ±
203
480 ± 110
Butyrate
420 ± 67
467 ±
177"
460 ±
Palmitate
249 + 84
429 ±
179 m
370 ± 80
50
Butyrate + proplonate
1003 + 159"*
161a
± 472**
20~0 + 350**
PalMitate + proplonate
1937
1197
+ 319 j
2420 ~ 440 I*
+ 371 j*
Hepatocytes (~10mg dry wt. cells) were incubated as described in the Materials and Methods section except that the duration of the incubation was 30 rain. Propionate and butyrate were present at 6 mM and palmitate was present along with carnitine at 1 mM. Results significantly different from propionate alone are expressed by *P < 0.05, **P < 0.01, n=5.
562
ANNE FAULKNER a n d HELEN T. POLLOCK Table 6. Effect of ~-cyanohydroxycinnamic acid on lactate plus pyruvate production in ovine hepatocytes Lactate
+ pyr,~vate p r o d u c t i o n
( u m o l / g dry wt. Control
No 8 u b s t r a t e
121
+ 11
Propionate
!5g
t 14.
74
~ 9**
Butyrate Butyrate
+ proplonate
Palmitate
75
+ 6 *w
121
t
,Cinnamlc a c i d
(38
(I
~ 5)
~ 10)
17
Palmltate
+ propionate
161
+ 11"
Palmltate
+ carnltlne
gq
! 13"
Palmltate
+ carnitlne + propionate
107 ~
,eltsl2hr)
26!
~ 2 2 '~ ( 2 8
108
~ 27 ~1
88 1q6
(40
B (8
*
, 13)
9)
" 22 ~'' *
'
1R)
(-20
'
2 ")
"~1
2P6
21
134
10 ~
lq~
17 ~
(30
(-1
*
1A~
'
8)
Hepatocytes were incubated as described in the Materials and Methods section. Propionate and butyrate were present at a concentration of 3 raM, palmitate and carnitine at 1 mM and ~-cyanohydroxycinnamic acid at 0.3 raM. Net rates of lactate and pyruvate production from propionate are given in parentheses. Results significantly different from equivalent incubations without added substrate are expressed by *P < 0.05. **P < 0.01, ***P < 0.001, n = 5.
production in the presence and absence of ~-cyanohydroxycinnamic acid reflects the amount normally transported and metabolized in the mitochondria, butyrate and palmitate also decreased pyruvate utilization. However, there was no detectable effect of butyrate or palmitate on the conversion of [1-]4C]lactate to ~4CO2 (lactate alone, 25.9 + 4.2; with butyrate, 25.9 -+ 5.0; with palmitate and carnitine 27.4_+6.1, n =5). The ability of palmitate to exert this effect on lactate and pyruvate production is dependent on the presence of exogenous carnitine (Table 6) suggesting that mitochondrial metabolism of this fatty acid is required.
DISCUSSION
Propionate taken up by ovine hepatocytes was used for oxidation, gluconeogenesis and lactate and pyruvate production. The fatty acids, butyrate and palmitate, reduced propionate uptake significantly. It is probable that this inhibition of propionate uptake by free fatty acids is due to competition for CoA within the cell, as all three compounds are initially metabolized to CoA esters. If availability of CoA within the mitochondria is a regulating factor, then the relative proportions of the long-chain and volatile fatty acids in the portal vein will greatly influence hepatic propionate uptake. Studies on the isolated enzyme propionyl-CoA synthetase from ruminant liver have shown that butyrate only slightly inhibits activity (Ash and Baird, 1973). The rate of propionate uptake observed in these hepatocytes is similar to or slightly in excess of rates of propionate uptake determined in vivo (Bergman, 1973), but the extracellular concentration is higher for the isolated hepatocytes. Butyrate and palmitate also changed the proportion of propionate metabolized by the various pathways. Both decreased the proportion metabolized to
CO2, lactate and pyruvate, but palmitate increased incorporation into glucose. The shift from CO2 production indicates reduced loss of propionate carbon in the tricarboxylic acid cycle which, for palmitate, was accompanied by an increased incorporation into glucose. Stimulation of gluconeogenesis by oleate in rat liver has been observed (Ross et al., 1967; Williamson et al., 1969) and is thought to result from both a stimulation of pyruvate carboxylase by increased acetyl CoA concentrations in the mitochondria and a decrease in cytosolic redox (see Table 3). However the extent of the stimulation in the sheep is small compared with that seen in the rat liver. The reduction in CO2 production from propionate may reflect a mechanism for sparing propionate carbon as other energy sources become available. The increased concentrations of the tricarboxylic acid cycle intermediate (especially malate which probably reflects increased oxaloacetate concentrations) should result in an increased capacity for acetyl CoA oxidation within the mitochondria. Stimulation of fatty acid oxidation by propionate has been observed in ovine hepatocytes (Lomax et al., 19841. Gluconeogenesis in the hepatocytes was determined by following incorporation of radioactivity from propionate and bicarbonate. The conversion of [l-14C]propionate (and [14C]NaHCO3) to [14C]glucose underestimates the amount of propionate converted to glucose by at least 50% (as indicated from the comparison of glucose production from [I-~4C] and [2-14C]propionate). The label from [l-14C]propionate is distributed evenly between CI and 4 of succinate and hence oxaloacetate during metabolism, the C4 being lost subsequently as CO2 on the formation of phosphoenolpyruvate. Taking this into account at least 30-40% of the propionate carbon is metabolized to glucose in the ovine hepatocytes. These values compare favourably with data obtained in vivo, where values of 40-50% are observed (Bergman et al., 1966). Similar values for net glucose production
Propionate metabolism in ovine hepatocytes
563
by ovine hepatocytes in vitro have been reported REFERENCES (Donaldson et al., 1979). Ash R. and Baird G. D. (1973) Activation of volatile fatty The ability of these fatty acids to decrease the rate acids in bovine liver and rumen epithelium. Evidence for of lactate and pyruvate production was observed control by autoregulation. Biochem. J. 136, 311-319. both in the presence of propionate and in its absence, Bergman E. N. (1973) Glucose metabolism in ruminants as related to hypoglycemiaand ketosis. Cornell Veterinarian when it was assumed that glycogen breakdown and 63, 341-382. subsequent metabolism of glucose via glycolysis was the source. The mitochondrial pyruvate transport Bergman E. N. and Wolff J. E. (1971) Metabolism of volatile fatty acids by liver and portal-drained viscera in inhibitor, ~-cyanohydroxycinnamic acid (Halestrap sheep. Am. J. Physiol. 221, 586-592. and Denton, 1974), was used to demonstrate that the Bergman E. N., Roe W. E. and Kon K. (1966) Quantitative effect of the fatty acids was to decrease the rate of aspects of propionate metabolism and gluconeogenesisin production of lactate and pyruvate from both endosheep. Am. J. PhysioL 211, 793-799. genous substrate and propionate rather than to stim- Bergmeyer H. U. (1974) In Methods o f Enzymatic Analysis. Academic Press, New York. ulate utilization; in fact there was some indication of reduced utilization. Inhibitory effects of long-chain Clark M. G., Filsell O, H. and Jarrett 1. G. (1976) Gluconeogenesis in isolated intact lamb liver cells. Effects of fatty acids on glucose utilization have been observed glucagon and butyrate. Biochem. J. 156, 671-680. in muscle and heart (Randle et al., 1964; Rennie and Holloszy, 1977), and in the liver have been attributed Demaugre F., Buc H. A., Cepanec C., Moncion A. and Leroux J. P. (1984) Influence of oleate oxidation to inhibition of phosphofructokinase by increased on pyruvate production and utilization in hepatocytes intracellular citrate concentrations (Siess et al., 1978). isolated from fed rats. Biochem. J. 222, 343-350. In rat hepatocytes mitochondrial utilization of pyru- Donaldson I. A., Lomax M. A. and Pogson C. I. (1979) vate was enhanced by oleate, so that the effects of The viability and glucagon responsiveness of isolated hepatocytes from adult sheep. Proc. Nutr. Soc. 38, 88A. fatty acids on lactate and pyruvate production in rat hepatocytes appears to be a combination of Halestrap A. P. and Denton R. M. (1974) Specificinhibition of pyruvate transport in rat liver mitochondria and decreased production and increased utilization human erythrocytes by ct-cyano-4-hydroxycinnamate. (Demaugre et al., 1984). Analysis of the carboxylic Biochem. J. 138, 313-316. acids present in ovine hepatocytes showed significant Jones G. B. (1965) Determination of the specific activity of increases in citrate after incubation with propionate labelled blood glucose by liquid scintillation using glucose and either butyrate or palmitate. Accumulation of pentaacetate. Anal. Biochem. 12, 249-258. citrate could inhibit glycolysis at the phosphofruc- Katz M. L. and Bergman E. N. (1969) Hepatic and portal tokinase step, lowering intracellular concentrations metabolism of glucose, free fatty acids, and ketone bodies in the sheep. Am. J. Physiol. 216, 953-960. of fructose 1,6-diphosphate and hence decreasing the activity of pyruvate kinase. This may explain Leng R. A. (1970) Glucose synthesis in ruminants. Adv. Vet. Sci. 14, 209260. inhibition of lactate and pyruvate production by Lomax M. A., Donaldson I. A. and Pogson C. I. (1984) The butyrate and palmitate from propionate, but it is control of fatty acid metabolism in liver cells from fed and unlikely that a similar mechanism could operate for starved sheep. Biochem. J. 214, 553-560. the inhibition from endogenous substrate as no Randle P. J., Newsholme E. A. and Garland P. B. (1964) increases in citrate concentrations were detected Regulation of glucose uptake by muscle. Effects of fatty when ovine hepatocytes were incubated with either acids, ketone bodies and pyruvate, and alloxan-diabetes butyrate or palmitate in the absence of propionate. In and starvation, on the uptake and metabolic fate of glucose in rat heart and diaphram muscles. Biochem. J. rat hepatocytes oleate oxidation increases citrate 93, 652~565. concentrations in whole cells (Demaugre et al., 1984). It can also be deduced that inhibition is not due to Rennie M. J. and Holloszy J. O. (1977) Inhibition of glucose uptake and glycogenolysis by availability of oleate in the presence of increased cytosolic concentrations of well-oxygenated perfused skeletal muscle. Biochem. J. CoA esters, as exogenous carnitine was needed for 168, 161-170. palmitate to show its full effect. Therefore some Ross B. D., Hems R. A., Freedland R. A. and Krebs H. A. mitochondrial metabolism of the long-chain free fatty (1967) Carbohydrate metabolism of the perfused rat liver. acids must be required to produce the inhibition of Biochem. J. 105, 869-875. lactate and pyruvate production. Siess E. A., Brocks D. G. and Wieland O. H. (1978) The present paper has demonstrated that butyrate Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated and palmitate can regulate the metabolism of profrom fed rats. Hoppe-Seyler's Z. Physiol. Chem. 359, pionate in ovine hepatocytes. The effect of both 785-798. appears to be to reduce propionate utilization and to Whitelaw E. and Williamson D. H. (1977) Effects of divert propionate metabolism away from oxidation lactation on ketogenesis from oleate or butyrate in rat and lactate and pyruvate production, and with the hepatocytes. Biochem. J. 164, 521-528. long-chain fatty acids, allowing increased incorpor- Williamson J. R., Browning E. T. and Scholz R. (1969) ation into glucose. Such mechanisms may produce Control of mechanisms of gluconeogenesis and ketomore efficient propionate utilization during periods genesis. Effects of oleate on gluconeogenesis in perfused of increased free fatty acid availability. rat liver. J. biol. Chem. 244, 4607-4616.
0305-0491/86 $3.00+ 0.00 PergamonJournals Ltd
P R O P I O N A T E M E T A B O L I S M A N D ITS R E G U L A T I O N BY F A T T Y ACIDS IN O V I N E H E P A T O C Y T E S ANNE FAULKNER* and HELEN T. POLLOCK Hannah Research Institute, Ayr KA6 5HL, Scotland (Tel: 0292-76013) (Received 7 November 1985)
Abstract--1. Propionate metabolism was studied in ovine hepatocytes. 2. The main products of metabolism were CO2, glucose, L-lactate and pyruvate. 3. The fatty acids, butyrate and palmitate inhibited propionate oxidation; butyrate inhibited but palmitate slightly stimulated gluconeogenesis from propionate. 4. Butyrate and palmitate also inhibited lactate and pyruvate production from both endogenous substrates and from propionate.
Propionate is produced by fermentation in the rumen and is a major precursor for glucose synthesis in the ruminant liver (Bergman, 1973). Over 90% of the propionate transported via the portal vein is extracted by the liver and it has been estimated that glucose synthesis from propionate could account for 50-60% of the glucose produced in the ruminant (Leng, 1970; Bergman, 1973). The pathway for glucose synthesis from propionate is well-established but little is known of the control mechanisms which regulate this pathway in the ruminant liver. As the ruminant absorbs little glucose from the diet, relying almost exclusively on gluconeogenesis for its glucose supply, rates of glucose synthesis are highest in the fed animal, decreasing during fasting (Bergman, 1973). This contrasts with the non-ruminant, where fasting stimulates gluconeogenesis. Hence the factors regulating gluconeogenesis in ruminant and non-ruminant species may be expected to differ markedly. The present paper describes studies on propionate metabolism in ovine hepatocytes and its regulation by the fatty acids, butyrate and palmitate, both of which are taken up by the sheep liver in vivo (Bergman and Wolff, 1971; Katz and Bergman, 1969).
Amersham International, Aylesbury, Bucks. Chemicals were from BDH Chemicals, Dagenham, Essex. Methods Hepatocytes were prepared by perfusing the caudate lobe with a collagenase solution (0.5% w/v) as previously described (Clark et al., 1976). Hepatocytes (~5mg dry wt) were incubated in a final volume of 2.5 ml of Krebs Ringer bicarbonate buffer gassed with 95% 02 and 5% CO2 containing 1.5% (w/v) gelatin and 1% (w/v) fatty-acid free albumin and incubated for 2 hr with shaking at 37°C. Reactions were stopped by the addition of HC1 04. Glucose, lactate, pyruvate, citrate, 2-oxoglutarate and malate were determined by enzymatic analysis (see Bergmeyer, 1973). Radioactive glucose was isolated and determined as the pentacetate (Jones, 1965). Production of ~4CO2 was determined by trapping CO2 in hyamine hydroxide following acidification of the incubation medium (Whitelaw and Williamson, 1977). Radioactivity at 0 min was determined routinely and subtracted from experimental values. Propionate was determined by glc. Statistical methods Results are expressed as means+SEM. Statistical analysis was performed using Student's t-test for paired observations.
RESULTS MATERIALS AND METHODS Animals Sheep were Finn x Dorset Horn cross-breeds. They were 1-4 years old and were fed hay ad lib plus a cereal mix (300400 g/day depending on age). A catheter was placed in the jugular vein on the day before the experiment, the sheep were then anaesthetized with an intravenous injection of sodium pentabarbitone before the caudate lobe of the liver was removed. Animals were killed before they recovered from anaesthesia. Materials Enzymes and biochemicals were obtained from Boehringer Corp., Lewes, Sussex and Sigma Chemical Co., Poole, Dorset. Radiochemicals were obtained from
*Address for correspondence: Department of Lactational Physiology and Biochemistry, Hannah Research Institute, Ayr, Scotland.
Metabolism o f propionate in ovine hepatocytes
Substantial quantities of [1-J4C]propionate were taken up by ovine hepatocytes and, following incubation, most of the radioactivity from the metabolized propionate was recovered in glucose and CO 2 (Table 1). In a second experiment, radioactive glucose production was determined using 14[C]NaHCO3 and [2-14C]propionate as well as [1-14C]propionate. Rates of radioactive glucose formation from [14C]NaHCO3 were similar to those from [1-~4C]propionate, but those from [2-~4C]propionate were more than double (Table 2). This is to be predicted as radioactivity from [l-14C]propionate and [14C]NaHCO3 equilibrate on the formation of succinate, and half of this radioactivity is lost subsequently at the phosphoenol pyruvate carboxykinase reaction. The radioactive CO2 lost at this step (equivalent to that incorporated into glucose for [1-14C]propionate) was subtracted from
559
560
ANNE FAULKNER and HELEN T, POLLOCK Table 1. [I-~4C]Propionate metabolism and its regulation b v bukvratc and palmitate , mo]
[I-
Propionate
.
.
.
.
.
~'2] prc~pi~!n:l~o/K ,]rv Wt , nf.ll ''~r
+ ~llt,,,r, a t e
+ palmit=+,,
.?t~
Conversion ef [!-t"C] pPopionate to:
l~ro
494
[~C:]
g]ucose
~ ~4
'?~,
6/)
I~
:£,*
11~
I~m
Hepatocytes were incubated as described in Materials and Methods section. Propionate was present at a concentration of 3 raM, butyrate at 3 mM and palmitate at 1 raM. Incubations containing palmitate also contained I mM carnitine. Results significantly differen t from propionate alone are expressed by *P <0.05. **P < 0.01, ***P < 0.001, n - 12.
the total ~4CO2 production to give values for the net production of CO 2 from [l-14C]propionate (Table 2).
proportion metabolized to [~4C]glucose (14% control. 15% butyrate, 22% palmitate).
Effects of Jree fatty acids on propionate metabolism The fatty acids butyratc and palmitate reduced significantly the uptake of propionate by the hepatocytes (Table 1), and the total and net J4CO2 production from [1-14C]propionate (Tables 1 and 2). Production of J4CO2 from [2-14C]propionate was also decreased slightly but not significantly (Table 2). Incorporation of radioactivity into glucose from all three precursors was significantly reduced by butyrate (Tables 1 and 2); palmitate significantly increased incorporation (Tables 1 and 2). The proportion of the [1-~4C]propionate taken up by the hepatocytes which was metabolized to ~4CO2 and glucose was also affected by free fatty acids. The proportion oxidized to 14CO2 decreased from 75% in the control to 64 and 62% in the presence of butyrate and palmitate respectively; palmitate but not butyrate increased the
Lactate and pyruuate production in ovine hepatocytes Lactate and pyruvatc were produced in ovine hepatocytes incubated without exogenous substrate (Table 3), presumably originating from breakdown and glycolysis of glycogen. The addition of propionate increased the amount of lactate and pyruvate produced (Table 3) and decreased the ratio of lactate to pyruvate (Table 4). The presence of butyrate or palmitate significantly reduced the amount of lactate and pyruvate produced by the hepatocytes both from endogenous substrate and the net amount produced from propionate (Table 3). The fatty acids had no significant effect on the ratio of lactate to pyruvate in the absence of propionate but increased the ratio in the presence of propionate (Table 4). The production of glucose, CO2, lactate and pyruvate accounted for 103, 87 and 92% of the propionate
Table 2. Glucose and (:02 production from radioactive precursors Hmol
suhstrate
Proplona~e
ineorporateJ/g
dr}' wt, cell/>hr
÷ butvrate
+ pilmit
~te
alone [I"C]
glucose
from:
[I-I"C] peopionate
l~a
[2-i"C]
~6% t 80
proptonate
[I.C]NaHCO
18%
' 2?
, %%
l!6
!q**i
15<
!~3F 12 c:
16"*
%8**
I):
t"C02 from: [1-t~'C] propionate
%QQ " 8~
27b
[2-I"C] propionate
38 + 13
~2
Net
i"CO
[I-I"c]
6?* 10
from:
pPopionate
246
~ 65
1 ~1
35*
Hepatocytes were incubated as described in Materials and Methods section. Propionate was present at a concentration of 5 raM, butyrate at 3 mM and palmitate at 1 mM. Incubations containing palmitate also contained l m M carnitine. Nel ~4CO2 production from [IJ4C]propionate was calculated as the difference between total ~4CO2 production and ~4C incorporation into glucose. Results significantly different from propionate alone are expressed by *P <0.05, **P <0.01, ***P < 0.001, n -= 7.
Propionate metabolism in ovine hepatocytes
561
Table 3. Effect of butyrate and palmitatc on lactate and pyruvate production Lactate plus pyruvate production (~mol/g dry wt. cells/ 2hr)
÷ butyrate
Endogenous production
Net production from propionate
÷ palmltate
19~ ± 25
47 ± 7"*
79 ± 27*
86 ± 35
33 ± 11"
~3 ± 16"
Hepatocytes were incubated as described in the Materials and Methods section. Propionate was present at 3mM, butyrate at 3mM and palmitate at l mM. Carnitine (l mM) was included in incubations containing palmitate. Endogenous production is that produced in the absence of propionate. Net production from propionate was calculated as the difference between total lactate and pyruvate production in the presence and absence of propionate. Results significantlydifferent from rates in the absence of butyrate or palmitate are expressed by *P < 0.05, **P < 0.01, n = 12. t a k e n u p by the hepatocytes in the absence o f free fatty acid, the presence o f b u t y r a t e or palmitat¢ respectively (Tables 1 a n d 3).
determine w h e t h e r the effects o f free fatty acids o n lactate a n d pyruvat¢ p r o d u c t i o n were due to decreased rates of p r o d u c t i o n or increased rates o f utilization. As m i g h t be predicted a - c y a n o h y d r o x y cinnamic acid significantly ( P < 0.01) increased the a m o u n t o f lactate a n d p y r u v a t e p r o d u c e d by ovine hepatocytes in all i n c u b a t i o n s (Table 6), indicating t h a t some lactate a n d pyruvat¢ p r o d u c e d was further metabolized normally. Butyrate a n d palmitate reduced lactate a n d p y r u v a t e p r o d u c t i o n f r o m e n d o g e n o u s substrate a n d a l m o s t eliminated the net p r o d u c t i o n f r o m p r o p i o n a t ¢ in b o t h the presence a n d absence o f the inhibitor (Table 6). This indicates t h a t there is a n effect o f these free fatty acids o n lactate a n d p y r u v a t e production. In addition, if the difference in the rate o f lactate a n d p y r u v a t e
Carboxylic acids in ovine hepatocytes after incubation W h e n hepatocytes were i n c u b a t e d with p r o p i o n a t e a n d either of the free fatty acids, the c o n c e n t r a t i o n s o f the carboxylic acids in the cells increased substantially (Table 5). T h e fatty acids alone did n o t produce this effect; p r o p i o n a t e p r o d u c e d increases in intracellular m a l a t e only (Table 5).
Effect of ct-cyanohydroxycinnamic acid on lactate and pyruvate production This i n h i b i t o r o f p y r u v a t e t r a n s p o r t to the mitoc h o n d r i a (Halestrap a n d D e n t o n , 1974) was used to
Table 4. Effect of butyrate and palmitate on the ratio oflactate to pyruvate produced by ovine hepatocytes Lactate
÷ butyrate
So addition
No substrate
Propionate
: pyruvate
+ palmitate
13.1
12.8
14.9
4.4
8.1
9.5
Hepatocytes were incubated as described in the Materials and Methods section. Propionate was present at 3 mM, bntyrate at 3 mM and palmitate at ! mM. Carnitine (1 mM) was included in incubations containing palmitate. Table 5. Concentrations of carboxylic acids in ovine hepatocytes Cohen.
(nmol/g dry wt. hepatoeytes)
2-oxoglutarate
malate
citrate
Proplonate
342 ± 89
861 ±
203
480 ± 110
Butyrate
420 ± 67
467 ±
177"
460 ±
Palmitate
249 + 84
429 ±
179 m
370 ± 80
50
Butyrate + proplonate
1003 + 159"*
161a
± 472**
20~0 + 350**
PalMitate + proplonate
1937
1197
+ 319 j
2420 ~ 440 I*
+ 371 j*
Hepatocytes (~10mg dry wt. cells) were incubated as described in the Materials and Methods section except that the duration of the incubation was 30 rain. Propionate and butyrate were present at 6 mM and palmitate was present along with carnitine at 1 mM. Results significantly different from propionate alone are expressed by *P < 0.05, **P < 0.01, n=5.
562
ANNE FAULKNER a n d HELEN T. POLLOCK Table 6. Effect of ~-cyanohydroxycinnamic acid on lactate plus pyruvate production in ovine hepatocytes Lactate
+ pyr,~vate p r o d u c t i o n
( u m o l / g dry wt. Control
No 8 u b s t r a t e
121
+ 11
Propionate
!5g
t 14.
74
~ 9**
Butyrate Butyrate
+ proplonate
Palmitate
75
+ 6 *w
121
t
,Cinnamlc a c i d
(38
(I
~ 5)
~ 10)
17
Palmltate
+ propionate
161
+ 11"
Palmltate
+ carnltlne
gq
! 13"
Palmltate
+ carnitlne + propionate
107 ~
,eltsl2hr)
26!
~ 2 2 '~ ( 2 8
108
~ 27 ~1
88 1q6
(40
B (8
*
, 13)
9)
" 22 ~'' *
'
1R)
(-20
'
2 ")
"~1
2P6
21
134
10 ~
lq~
17 ~
(30
(-1
*
1A~
'
8)
Hepatocytes were incubated as described in the Materials and Methods section. Propionate and butyrate were present at a concentration of 3 raM, palmitate and carnitine at 1 mM and ~-cyanohydroxycinnamic acid at 0.3 raM. Net rates of lactate and pyruvate production from propionate are given in parentheses. Results significantly different from equivalent incubations without added substrate are expressed by *P < 0.05. **P < 0.01, ***P < 0.001, n = 5.
production in the presence and absence of ~-cyanohydroxycinnamic acid reflects the amount normally transported and metabolized in the mitochondria, butyrate and palmitate also decreased pyruvate utilization. However, there was no detectable effect of butyrate or palmitate on the conversion of [1-]4C]lactate to ~4CO2 (lactate alone, 25.9 + 4.2; with butyrate, 25.9 -+ 5.0; with palmitate and carnitine 27.4_+6.1, n =5). The ability of palmitate to exert this effect on lactate and pyruvate production is dependent on the presence of exogenous carnitine (Table 6) suggesting that mitochondrial metabolism of this fatty acid is required.
DISCUSSION
Propionate taken up by ovine hepatocytes was used for oxidation, gluconeogenesis and lactate and pyruvate production. The fatty acids, butyrate and palmitate, reduced propionate uptake significantly. It is probable that this inhibition of propionate uptake by free fatty acids is due to competition for CoA within the cell, as all three compounds are initially metabolized to CoA esters. If availability of CoA within the mitochondria is a regulating factor, then the relative proportions of the long-chain and volatile fatty acids in the portal vein will greatly influence hepatic propionate uptake. Studies on the isolated enzyme propionyl-CoA synthetase from ruminant liver have shown that butyrate only slightly inhibits activity (Ash and Baird, 1973). The rate of propionate uptake observed in these hepatocytes is similar to or slightly in excess of rates of propionate uptake determined in vivo (Bergman, 1973), but the extracellular concentration is higher for the isolated hepatocytes. Butyrate and palmitate also changed the proportion of propionate metabolized by the various pathways. Both decreased the proportion metabolized to
CO2, lactate and pyruvate, but palmitate increased incorporation into glucose. The shift from CO2 production indicates reduced loss of propionate carbon in the tricarboxylic acid cycle which, for palmitate, was accompanied by an increased incorporation into glucose. Stimulation of gluconeogenesis by oleate in rat liver has been observed (Ross et al., 1967; Williamson et al., 1969) and is thought to result from both a stimulation of pyruvate carboxylase by increased acetyl CoA concentrations in the mitochondria and a decrease in cytosolic redox (see Table 3). However the extent of the stimulation in the sheep is small compared with that seen in the rat liver. The reduction in CO2 production from propionate may reflect a mechanism for sparing propionate carbon as other energy sources become available. The increased concentrations of the tricarboxylic acid cycle intermediate (especially malate which probably reflects increased oxaloacetate concentrations) should result in an increased capacity for acetyl CoA oxidation within the mitochondria. Stimulation of fatty acid oxidation by propionate has been observed in ovine hepatocytes (Lomax et al., 19841. Gluconeogenesis in the hepatocytes was determined by following incorporation of radioactivity from propionate and bicarbonate. The conversion of [l-14C]propionate (and [14C]NaHCO3) to [14C]glucose underestimates the amount of propionate converted to glucose by at least 50% (as indicated from the comparison of glucose production from [I-~4C] and [2-14C]propionate). The label from [l-14C]propionate is distributed evenly between CI and 4 of succinate and hence oxaloacetate during metabolism, the C4 being lost subsequently as CO2 on the formation of phosphoenolpyruvate. Taking this into account at least 30-40% of the propionate carbon is metabolized to glucose in the ovine hepatocytes. These values compare favourably with data obtained in vivo, where values of 40-50% are observed (Bergman et al., 1966). Similar values for net glucose production
Propionate metabolism in ovine hepatocytes
563
by ovine hepatocytes in vitro have been reported REFERENCES (Donaldson et al., 1979). Ash R. and Baird G. D. (1973) Activation of volatile fatty The ability of these fatty acids to decrease the rate acids in bovine liver and rumen epithelium. Evidence for of lactate and pyruvate production was observed control by autoregulation. Biochem. J. 136, 311-319. both in the presence of propionate and in its absence, Bergman E. N. (1973) Glucose metabolism in ruminants as related to hypoglycemiaand ketosis. Cornell Veterinarian when it was assumed that glycogen breakdown and 63, 341-382. subsequent metabolism of glucose via glycolysis was the source. The mitochondrial pyruvate transport Bergman E. N. and Wolff J. E. (1971) Metabolism of volatile fatty acids by liver and portal-drained viscera in inhibitor, ~-cyanohydroxycinnamic acid (Halestrap sheep. Am. J. Physiol. 221, 586-592. and Denton, 1974), was used to demonstrate that the Bergman E. N., Roe W. E. and Kon K. (1966) Quantitative effect of the fatty acids was to decrease the rate of aspects of propionate metabolism and gluconeogenesisin production of lactate and pyruvate from both endosheep. Am. J. PhysioL 211, 793-799. genous substrate and propionate rather than to stim- Bergmeyer H. U. (1974) In Methods o f Enzymatic Analysis. Academic Press, New York. ulate utilization; in fact there was some indication of reduced utilization. Inhibitory effects of long-chain Clark M. G., Filsell O, H. and Jarrett 1. G. (1976) Gluconeogenesis in isolated intact lamb liver cells. Effects of fatty acids on glucose utilization have been observed glucagon and butyrate. Biochem. J. 156, 671-680. in muscle and heart (Randle et al., 1964; Rennie and Holloszy, 1977), and in the liver have been attributed Demaugre F., Buc H. A., Cepanec C., Moncion A. and Leroux J. P. (1984) Influence of oleate oxidation to inhibition of phosphofructokinase by increased on pyruvate production and utilization in hepatocytes intracellular citrate concentrations (Siess et al., 1978). isolated from fed rats. Biochem. J. 222, 343-350. In rat hepatocytes mitochondrial utilization of pyru- Donaldson I. A., Lomax M. A. and Pogson C. I. (1979) vate was enhanced by oleate, so that the effects of The viability and glucagon responsiveness of isolated hepatocytes from adult sheep. Proc. Nutr. Soc. 38, 88A. fatty acids on lactate and pyruvate production in rat hepatocytes appears to be a combination of Halestrap A. P. and Denton R. M. (1974) Specificinhibition of pyruvate transport in rat liver mitochondria and decreased production and increased utilization human erythrocytes by ct-cyano-4-hydroxycinnamate. (Demaugre et al., 1984). Analysis of the carboxylic Biochem. J. 138, 313-316. acids present in ovine hepatocytes showed significant Jones G. B. (1965) Determination of the specific activity of increases in citrate after incubation with propionate labelled blood glucose by liquid scintillation using glucose and either butyrate or palmitate. Accumulation of pentaacetate. Anal. Biochem. 12, 249-258. citrate could inhibit glycolysis at the phosphofruc- Katz M. L. and Bergman E. N. (1969) Hepatic and portal tokinase step, lowering intracellular concentrations metabolism of glucose, free fatty acids, and ketone bodies in the sheep. Am. J. Physiol. 216, 953-960. of fructose 1,6-diphosphate and hence decreasing the activity of pyruvate kinase. This may explain Leng R. A. (1970) Glucose synthesis in ruminants. Adv. Vet. Sci. 14, 209260. inhibition of lactate and pyruvate production by Lomax M. A., Donaldson I. A. and Pogson C. I. (1984) The butyrate and palmitate from propionate, but it is control of fatty acid metabolism in liver cells from fed and unlikely that a similar mechanism could operate for starved sheep. Biochem. J. 214, 553-560. the inhibition from endogenous substrate as no Randle P. J., Newsholme E. A. and Garland P. B. (1964) increases in citrate concentrations were detected Regulation of glucose uptake by muscle. Effects of fatty when ovine hepatocytes were incubated with either acids, ketone bodies and pyruvate, and alloxan-diabetes butyrate or palmitate in the absence of propionate. In and starvation, on the uptake and metabolic fate of glucose in rat heart and diaphram muscles. Biochem. J. rat hepatocytes oleate oxidation increases citrate 93, 652~565. concentrations in whole cells (Demaugre et al., 1984). It can also be deduced that inhibition is not due to Rennie M. J. and Holloszy J. O. (1977) Inhibition of glucose uptake and glycogenolysis by availability of oleate in the presence of increased cytosolic concentrations of well-oxygenated perfused skeletal muscle. Biochem. J. CoA esters, as exogenous carnitine was needed for 168, 161-170. palmitate to show its full effect. Therefore some Ross B. D., Hems R. A., Freedland R. A. and Krebs H. A. mitochondrial metabolism of the long-chain free fatty (1967) Carbohydrate metabolism of the perfused rat liver. acids must be required to produce the inhibition of Biochem. J. 105, 869-875. lactate and pyruvate production. Siess E. A., Brocks D. G. and Wieland O. H. (1978) The present paper has demonstrated that butyrate Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated and palmitate can regulate the metabolism of profrom fed rats. Hoppe-Seyler's Z. Physiol. Chem. 359, pionate in ovine hepatocytes. The effect of both 785-798. appears to be to reduce propionate utilization and to Whitelaw E. and Williamson D. H. (1977) Effects of divert propionate metabolism away from oxidation lactation on ketogenesis from oleate or butyrate in rat and lactate and pyruvate production, and with the hepatocytes. Biochem. J. 164, 521-528. long-chain fatty acids, allowing increased incorpor- Williamson J. R., Browning E. T. and Scholz R. (1969) ation into glucose. Such mechanisms may produce Control of mechanisms of gluconeogenesis and ketomore efficient propionate utilization during periods genesis. Effects of oleate on gluconeogenesis in perfused of increased free fatty acid availability. rat liver. J. biol. Chem. 244, 4607-4616.