Gluconeogenic Dependence on Ketogenesis in Isolated Sheep Hepatocytes1 JESSE C. CHOW, CHERYL PLANCK-MEYER, and BARRY W. JESSE Department of Animal Sciences Rutgers, The Slale University New Brunswick, NJ 08903 ABSTRACT
INTRODUCTION
Dependence of gluconeogenesis on ~ oxidation and ketogenesis from longchain fatty acids was examined in isolated sheep hepatocytes. Hepatocytes were incubated with a combination of gluconeogenic precursors (2 mM pyruvate, 20 mM lactate, and 5 mM propionate) plus other fatty acids, in the presence and absence of telradecylglycidic acid, an inhibitor of the carnitine palmitoyltransferase reaction. Palmitate oxidation to total acid-soluble metabolites or ~-hydroxybutyrate was markedly inhibited by the addition of tetradecylglycidic acid. In general, oxidation of palmitate to carbon dioxide was not altered by telI"adecylglycidic acid. Glucose production was inhibited 28 to 50% in the presence of tetradecylglycidic acid. Addition of acetate and butyrate inhibited gluconeogenesis, but octanoate addition had a slight stimulatory effect In the presence of tetradecylglycidic acid, butyrate, but not acetate, addition further reduced gluconeogenesis. In contrast, addition of octanoate in the presence of tetradecylglycidic acid restored gluconeogenic rates to control values. The results are consistent with observations in several nonruminant species and suggest that, as in those species, ruminant gluconeogenesis requires at least a basal rate of ~-oxida tion and ketogenesis from long-chain fatty acids to support maximum gluconeogenic rates. (Key words: gluconeogenesis, ketogenesis, hepatocytes)
Ruminants are in a state of continuous gluconeogenesis due to the degradation of most of the dietary carbohydrate to VFA in the rumen fermentation (23). In nonruminants, an association between hepatic gluconeogenesis and ketogenesis has long been observed with both processes operating in concert under a variety of metabolic conditions (18). Maximal rates of gluconeogenesis are generally not observed in the absence of ketogenesis in the liver of the nonruminant (10). A similar relationship seems to operate in ruminant liver: continuous gluconeogenesis is accompanied by a continuous hepatic ketogenesis (12). Various researchers have examined the relationship between hepatic ketogenesis and gluconeogenesis in nonruminants using a variety of metabolic inhibitors, most notably tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyltransferase I [CPT I; EC 2.3.1.21; (8)]. Inhibition of CPT I blocks transport of long-chain fatty acids (LCFA) into the mitochondria, thereby preventing ~-oxidation and ketogenesis from LCFA (16). Blocking LCFA oxidation with TDGA has resulted in the inhibition of gluconeogenesis in the livers of animals from several different species, both in vivo and in vitro, including the rat, dog, and guinea pig (20, 21). However, the dependence of gluconeogenesis upon an active ketogenesis in ruminant liver has not been ascertained. Hepatic ketogenesis is a tightly regulated process in ruminants and nonruminants alike (e.g., 13, 16). One aspect of this regulation in the ruminant is the ability of VFA, such as acetate and propionate from the rumen fermentation, to inhibit LCFA oxidation and ketogenesis (13). Short-chain fatty acids, such as acetate and butyrate, can undergo activation and metabolism in ruminant liver, although acetate metabolism occurs at a relatively low rate (4, 17). Thus, although ketogenesis may be re-
Received February 6, 1989. Accepted October 5. 1989. lSupported in part by Rutgers Research Council Grant Number 2-02262 and the New Jersey Agricultural Experiment Station.
1990 J Dairy Sci 73:683-689
683
684
CHOW ET AL.
treme cases as much as 120 min of collagenase perfusion was needed. Hepatocytes were washed three times by gentle centrifugation at 60 x g followed by resuspension in wash buffer (same composition as perfusion buffer but without EGTA). After the final wash, hepatocytes were resuspended to an approximate final volume for counting and viability estimates by trypan blue staining. The final volume of the cell suspension was adjusted based on cell counts. In vitro incubations were conducted as described by Forsell et al. (11) and Jesse et al. (13). Incubations were initiated by the addition of .5 ml of hepatocyte suspension, corresponding to 2 to 10 mg of viable dry cell weight, to 25-ml Erlenmeyer flasks containing 2.5 ml of media. Media were composed of Krebs-Ringer MATERIALS AND METHODS bicarbonate buffer, pH 7.4, containing 25 mM HEPES, 2 mM dl-carnitine, .2 mM dibutyryl Meterials cyclic AMP, and .25 mM bovine serum albuTetradecylglycidic acid (McN-3802-21-98) min and supplemented with fatty acids and was a generous gift from McNeil Pharmaceuti- gluconeogenic substrates at concentrations cals, Inc. (Spring House, PA). Type 1 collagen- noted in the text. Palmitate and TDGA were ase was obtained from Worthington Biochemi- complexed with bovine serum albumin before cals, Inc. (Freehold, NJ), 1-[14C]palmitic acid addition to the incubation flasks by gentle heatwas from New England Nuclear (Boston, MA), ing (5ST) of an albumin solution to which the and all other enzymes and reagents were from palmitate or TOGA had been added. IncubaSigma Chemical Co. (St. Louis, MO). tions were conducted at 37'C and were terminated by the addition of .2 ml of concentrated perchIoric acid. When flasks contained 1Methods [14C]palmitate, .3 ml of hyamine hydroxide Hepatocytes were isolated from the caudate was added to suspended center wells, and the lobe of the liver from 12- to 32-wk-old fed incubations continued for an additional 60 min Dorset ram lambs. The procedure was deto trap 14COZ . Media were neutralized with scribed by Forsell et al. (11). Briefly, the caudate lobe was removed immediately after potassium carbonate, and an aliquot was immeslaughter and perfused through the major blood diately removed for counting of total [14C]acidvessels with ice-cold Ca-free perfusion buffer soluble metabolites (ASM) when 1-[l4C]palmi[10 mM N-2-Hydroxyethylpiperazine-N'-2- tate was included. Remaining media were ethanesulfonic acid (HEPES), pH 7.4, 140 roM stored frozen until analyzed for glucose and ~ NaCI, 7.1 mM KCI, .1 mM ethyleneglycol-bis- hydroxybutyrate (BHBA). ~-Hydroxybutyrate was detennined enzymatically by the method of (~-aminoethyl ether)-tetraacetic acid (EGTA)]. Following transport to the laboratory, the lobe Williamson and Mellanby (22). Glucose was was first perfused with warm perfusion buffer, measured using a commercially available kit followed by a recirculating perfusion of colla- (Sigma, Number 510), with the procedure genase solution (perfusion buffer containing I scaled down to enable the reactions to be run in mg/ml Type 1 collagenase and 5 mM CaCI2) at 96-well microtiter plates, and the subsequent 37"C. The time between slaughter and the start use of a microtiter plate reader (Model Number of the collagenase perfusion was 10 to 15 min. EL309, Biotek Instruments, Inc., Winooski, Generally 30 to 60 min of perfusion with the VT) to measure absorbance. Cell dry weights collagenase solution was required for complete were determined by drying aliquots of hepatodissociation of the lobe, although in some ex- cytes and correcting for the salt content of an
quired to SUpport gluconeogenesis, the liver may not discriminate between short- or longchain fatty acids as a source of acetyl-CoA for ketogenesis. Such a situation in the fed state would allow VFA from the rumen to serve the dual role of limiting LCFA oxidation while at the same time maintaining a pennissive effect on hepatic gluconeogenesis. The objectives of this research were 1) to detennine if inhibition of ketogenesis from LCFA by TDGA would result in a concurrent inhibition of gluconeogenesis in isolated sheep hepatocytes, and if so, 2) to detennine if addition of VFA (acetate and butyrate) would overcome that inhibition. Preliminary reports of this research have appeared (6, 14).
Journal of Dairy Science Vol. 73.
No.3. 1990
685
HEPATOCYTE GLUCONEOOENESIS AND KETOGENESIS
TABLE I. Palmitate oxidation and ketogenesis by isolated sheep hepatocytes in the presence and 'absence of acetate, butyrate, and tetradecylglycidic acid (TOGA). [14C)Palmitate oxidation to ( 14C]Acid soluble
Additions
Palmitate
+ Palmitale + acetate
+
Palmitate + butyrate
+
~·Hydroxybutyrate
14C02
Metabolites
TOGA
production
- - - (pmoVrng per min) - - -
-
X
X
SE 214 8 47 lOS b 29 208 8 48 97.6b 24.S 193 8 38 76.cj> 16.3
X
SE 49.68 11.8 53.38 16.5 47.4 8 11.4 46.8 8 13.3 41.8b 8.5 29Ac 7.5
(nmoVrng per min) -
.264b .077c
.283 b .05
INTRODUCTION
Dependence of gluconeogenesis on ~ oxidation and ketogenesis from longchain fatty acids was examined in isolated sheep hepatocytes. Hepatocytes were incubated with a combination of gluconeogenic precursors (2 mM pyruvate, 20 mM lactate, and 5 mM propionate) plus other fatty acids, in the presence and absence of telradecylglycidic acid, an inhibitor of the carnitine palmitoyltransferase reaction. Palmitate oxidation to total acid-soluble metabolites or ~-hydroxybutyrate was markedly inhibited by the addition of tetradecylglycidic acid. In general, oxidation of palmitate to carbon dioxide was not altered by telI"adecylglycidic acid. Glucose production was inhibited 28 to 50% in the presence of tetradecylglycidic acid. Addition of acetate and butyrate inhibited gluconeogenesis, but octanoate addition had a slight stimulatory effect In the presence of tetradecylglycidic acid, butyrate, but not acetate, addition further reduced gluconeogenesis. In contrast, addition of octanoate in the presence of tetradecylglycidic acid restored gluconeogenic rates to control values. The results are consistent with observations in several nonruminant species and suggest that, as in those species, ruminant gluconeogenesis requires at least a basal rate of ~-oxida tion and ketogenesis from long-chain fatty acids to support maximum gluconeogenic rates. (Key words: gluconeogenesis, ketogenesis, hepatocytes)
Ruminants are in a state of continuous gluconeogenesis due to the degradation of most of the dietary carbohydrate to VFA in the rumen fermentation (23). In nonruminants, an association between hepatic gluconeogenesis and ketogenesis has long been observed with both processes operating in concert under a variety of metabolic conditions (18). Maximal rates of gluconeogenesis are generally not observed in the absence of ketogenesis in the liver of the nonruminant (10). A similar relationship seems to operate in ruminant liver: continuous gluconeogenesis is accompanied by a continuous hepatic ketogenesis (12). Various researchers have examined the relationship between hepatic ketogenesis and gluconeogenesis in nonruminants using a variety of metabolic inhibitors, most notably tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyltransferase I [CPT I; EC 2.3.1.21; (8)]. Inhibition of CPT I blocks transport of long-chain fatty acids (LCFA) into the mitochondria, thereby preventing ~-oxidation and ketogenesis from LCFA (16). Blocking LCFA oxidation with TDGA has resulted in the inhibition of gluconeogenesis in the livers of animals from several different species, both in vivo and in vitro, including the rat, dog, and guinea pig (20, 21). However, the dependence of gluconeogenesis upon an active ketogenesis in ruminant liver has not been ascertained. Hepatic ketogenesis is a tightly regulated process in ruminants and nonruminants alike (e.g., 13, 16). One aspect of this regulation in the ruminant is the ability of VFA, such as acetate and propionate from the rumen fermentation, to inhibit LCFA oxidation and ketogenesis (13). Short-chain fatty acids, such as acetate and butyrate, can undergo activation and metabolism in ruminant liver, although acetate metabolism occurs at a relatively low rate (4, 17). Thus, although ketogenesis may be re-
Received February 6, 1989. Accepted October 5. 1989. lSupported in part by Rutgers Research Council Grant Number 2-02262 and the New Jersey Agricultural Experiment Station.
1990 J Dairy Sci 73:683-689
683
684
CHOW ET AL.
treme cases as much as 120 min of collagenase perfusion was needed. Hepatocytes were washed three times by gentle centrifugation at 60 x g followed by resuspension in wash buffer (same composition as perfusion buffer but without EGTA). After the final wash, hepatocytes were resuspended to an approximate final volume for counting and viability estimates by trypan blue staining. The final volume of the cell suspension was adjusted based on cell counts. In vitro incubations were conducted as described by Forsell et al. (11) and Jesse et al. (13). Incubations were initiated by the addition of .5 ml of hepatocyte suspension, corresponding to 2 to 10 mg of viable dry cell weight, to 25-ml Erlenmeyer flasks containing 2.5 ml of media. Media were composed of Krebs-Ringer MATERIALS AND METHODS bicarbonate buffer, pH 7.4, containing 25 mM HEPES, 2 mM dl-carnitine, .2 mM dibutyryl Meterials cyclic AMP, and .25 mM bovine serum albuTetradecylglycidic acid (McN-3802-21-98) min and supplemented with fatty acids and was a generous gift from McNeil Pharmaceuti- gluconeogenic substrates at concentrations cals, Inc. (Spring House, PA). Type 1 collagen- noted in the text. Palmitate and TDGA were ase was obtained from Worthington Biochemi- complexed with bovine serum albumin before cals, Inc. (Freehold, NJ), 1-[14C]palmitic acid addition to the incubation flasks by gentle heatwas from New England Nuclear (Boston, MA), ing (5ST) of an albumin solution to which the and all other enzymes and reagents were from palmitate or TOGA had been added. IncubaSigma Chemical Co. (St. Louis, MO). tions were conducted at 37'C and were terminated by the addition of .2 ml of concentrated perchIoric acid. When flasks contained 1Methods [14C]palmitate, .3 ml of hyamine hydroxide Hepatocytes were isolated from the caudate was added to suspended center wells, and the lobe of the liver from 12- to 32-wk-old fed incubations continued for an additional 60 min Dorset ram lambs. The procedure was deto trap 14COZ . Media were neutralized with scribed by Forsell et al. (11). Briefly, the caudate lobe was removed immediately after potassium carbonate, and an aliquot was immeslaughter and perfused through the major blood diately removed for counting of total [14C]acidvessels with ice-cold Ca-free perfusion buffer soluble metabolites (ASM) when 1-[l4C]palmi[10 mM N-2-Hydroxyethylpiperazine-N'-2- tate was included. Remaining media were ethanesulfonic acid (HEPES), pH 7.4, 140 roM stored frozen until analyzed for glucose and ~ NaCI, 7.1 mM KCI, .1 mM ethyleneglycol-bis- hydroxybutyrate (BHBA). ~-Hydroxybutyrate was detennined enzymatically by the method of (~-aminoethyl ether)-tetraacetic acid (EGTA)]. Following transport to the laboratory, the lobe Williamson and Mellanby (22). Glucose was was first perfused with warm perfusion buffer, measured using a commercially available kit followed by a recirculating perfusion of colla- (Sigma, Number 510), with the procedure genase solution (perfusion buffer containing I scaled down to enable the reactions to be run in mg/ml Type 1 collagenase and 5 mM CaCI2) at 96-well microtiter plates, and the subsequent 37"C. The time between slaughter and the start use of a microtiter plate reader (Model Number of the collagenase perfusion was 10 to 15 min. EL309, Biotek Instruments, Inc., Winooski, Generally 30 to 60 min of perfusion with the VT) to measure absorbance. Cell dry weights collagenase solution was required for complete were determined by drying aliquots of hepatodissociation of the lobe, although in some ex- cytes and correcting for the salt content of an
quired to SUpport gluconeogenesis, the liver may not discriminate between short- or longchain fatty acids as a source of acetyl-CoA for ketogenesis. Such a situation in the fed state would allow VFA from the rumen to serve the dual role of limiting LCFA oxidation while at the same time maintaining a pennissive effect on hepatic gluconeogenesis. The objectives of this research were 1) to detennine if inhibition of ketogenesis from LCFA by TDGA would result in a concurrent inhibition of gluconeogenesis in isolated sheep hepatocytes, and if so, 2) to detennine if addition of VFA (acetate and butyrate) would overcome that inhibition. Preliminary reports of this research have appeared (6, 14).
Journal of Dairy Science Vol. 73.
No.3. 1990
685
HEPATOCYTE GLUCONEOOENESIS AND KETOGENESIS
TABLE I. Palmitate oxidation and ketogenesis by isolated sheep hepatocytes in the presence and 'absence of acetate, butyrate, and tetradecylglycidic acid (TOGA). [14C)Palmitate oxidation to ( 14C]Acid soluble
Additions
Palmitate
+ Palmitale + acetate
+
Palmitate + butyrate
+
~·Hydroxybutyrate
14C02
Metabolites
TOGA
production
- - - (pmoVrng per min) - - -
-
X
X
SE 214 8 47 lOS b 29 208 8 48 97.6b 24.S 193 8 38 76.cj> 16.3
X
SE 49.68 11.8 53.38 16.5 47.4 8 11.4 46.8 8 13.3 41.8b 8.5 29Ac 7.5
(nmoVrng per min) -
.264b .077c
.283 b .05
