Comp. Biochem. Physiol. Vol. 98B, No. 2/3, pp. 283-286, 1991 0305-0491/91 $3.00 + 0.00 Printed in Great Britain © 1991 Pergamon Press pie

EFFECT OF LACTATION ON GLUCONEOGENESIS A N D KETOGENESIS IN OVINE HEPATOCYTES

ANNE FAULKNER and HELEN T. POLLOCK

Hannah Research Institute, Ayr KA5 6HL, UK (Tel: 0292 76013)

(Received 2 August 1990)

Abstract--1. Rates of glucose synthesis from radioactive precursors and ketogenesis were determined in hepatocytes from control and lactating sheep.

2. Gluconeogenesis from propionate was the same in both groups. Gluconeogenesis from lac- tate + pyruvate was three-fold higher in hepatocytes from lactating sheep. Palmitate stimulated gluconeo- genesis from lactate + pyruvate in both groups.

3. Rates of ketogenesis from palmitate but not butyrate were slightly higher in hepatocytes from lactating sheep. No other differences in the metabolism of palmitate or butyrate were seen in the two groups. Exogenous carnitine stimulated ketogenesis from palmitate. Propionate inhibited ketogenesis from palmitate and butyrate. Lactate + pyruvate also inhibited ketogenesis slightly but stimulated oxidation and esterification.

4. It is concluded that the major changes in glucose and ketone production seen in the lactating ruminant are not the result of long-term changes within the hepatocyte but occur because of the changes in substrate supply to the liver and changes in intracellular concentrations of metabolites.

INTRODUCTION

The ruminant absorbs little glucose from the diet and relies almost exclusively on gluconeogenesis for its glucose supply (Leng, 1970; Bergman, 1973). During periods of high glucose requirement (e.g. pregnancy and lactation) the output from the liver is increased several-fold (Bergman and Hogue, 1967; Lomax et al., 1983) and it is at this time that animals become increasingly susceptible to ketosis (Bergman, 1971; Schultz, 1974; Baird, 1977). Despite the increased rates of gluconeogenesis in lactation, the activities of most of the hepatic enzymes (the exception is phos- phoenolpyruvate carboxykinase) involved do not ap- pear to change dramatically when rates of glucose synthesis go up (Mackie and Campbell, 1972; Smith and Walsh, 1982). In contrast, in the rat liver exper- imental conditions which result in enhanced rates of gluconeogenesis (starvation, hormone treatment, etc.) are accompanied by several-fold increases in the activities of the gluconeogenic enzymes (Ash- more and Weber, 1968) and the stimulation of gluco- neogenesis persists and can be detected in the isolated organ or cell (Ross et aL, 1967). In addition livers from rats at peak lactation have a lower capacity for ketogenesis (Whitelaw and Williamson, 1977).

This paper presents data from an investigation into the differences in the rates of gluconeogenesis and ketogenesis in hepatocytes isolated from lactating and non-lactating sheep, in order to identify intrinsic changes which occur in liver cells of the ruminant as a result of lactation.

MATERIALS AND METHODS

Sheep were Finn-Dorset Horn cross-breeds. They were I-4 years old and were fed bay ad libitum plus a cereal mix

(300--400g/day for non-lactating and 1200g/day for lactating animals). Sheep were anaesthetized with an intravenous injection of sodium pentabarbitone before the caudate lobe of the liver was removed. Animals were killed with an overdose of anaesthetic before regaining consciousness.

Hepatocytes were prepared by perfusing the caudate lobe with a collagenase containing solution (0.5%, w/v) as described previously (Clark et al., 1976; Faulkner, 1986; Faulkner and Pollock, 1990). Hepatocytes (about 5 nag dry wt) were incubated in a final volume of 2.5 ml Krebs Ringer bicarbonate containing 1.5% (w/v) gelatin, 1% (w/v) fatty acid-free albumin, radioactive precursors (0.5 #Ci NaHCO2; 0.05 #Ci propionate, butyrate and palmitate) and various additions as indicated in the Results section and incubated for 2 hr at 37°C with shaking. Incubations were stopped by the addition of 50 #1 10 N HC104 (Faulkner and Pollock, 1990).

Acetoacetate and fl-hydroxybutyrate were determined enzymatic.ally (Williamson et al., 1962). Rates of ketone production were calculated as the sum of acetoacetate and fl-hydroxybutyrate. Radioactive glucose was isolated and determined as the pentaacetate (Jones, 1965; Faulkner and Pollock, 1990). Production of t4CO2 was determined by trapping evolved CO2 in hyamine hydroxide following acidifcation of the medium (Whitelaw and WiUiamson, 1977). Palmitate uptake and esterification was determined by separating cells from medium after incubation and extracting each with chloroform/methanol (2:1, v/v) (Whitelaw and Williamson, 1977). Radioactivity extracted in the chloroform phase from cell pellets was used as a measure of esterification, and disappearance of radioactivity from the chloroform phase extracted from medium was used to calculate palmitate uptake. Analysis by thin-layer chromatography of the chloroform extracts confirmed that the esterified fat was retained in the hepatoeytes. Radioactivity in COz, glucose, palmitate and estefified fats at 0 hr was determined and subtracted from experimental values. Palmitate concentrations were determined by gel- layer chromatography.

283 CBP(B) 98-2/~3

284 ANNE FAULKNER and HELEN T. POLLOCK

Table 1. Gluconeogenesis in hepatocytes from control and lactating sheep

Radioactive Gluconeogenic precursor precursor Palmitate

14C precursor incorporation into glucose /~mol/mg dry wt/2 hr

Control Lactating

Propionate [l-14C]propionate - 123 ± 15 (10) 121 ± 19 (10) Propionate [l-14C]propionate + 146 ± 19 (10)* 144 ± 21 (10)* Propionate NaH14CO3 - 194 + 35 (5) 249 ± 49 (5) Propionate NaH14CO3 + 198 + 37 (5) 269 __. 51 (5) Lactate + pyruvate NaHI4CO3 - 29.8 ± 4.1 (5) 75.8 ± 10.1 (5)t Lactate + pyruvate NaHI4CO3 + 58.9 + 11.3 (5)** 117.1 ± 14.2 (5)~'**

Hepatocytes were incubated with propionate (3 mM) or L-lactate (3 mM) + pyruvate (0.3 mM) with the radioactive precursors shown in the presence or absence of palmitate (1 mM) + carnitine (1 mM). Rates of [t4C]glucose formation were determined as described in Materials and Methods section. Results are means 5- SE with the number of animals in parentheses. Values for lactating animals significantly different from control are ~-P < 0.05. Values obtained in the presence of palmitate significantly different from those without palmitat¢ are *P < 0.05, **P < 0.01.

RESULTS

Rates of gluconeogenesis f rom prop iona te were similar in hepatocytes f rom control and lactat ing sheep, bu t rates f rom lactate + pyruvate were signifi- cant ly higher in hepatocytes f rom lactat ing animals (Table 1). Glucose synthesis f rom b o t h p rop iona te and lactate + pyruvate was s t imulated significantly by palmi ta te bu t the s t imulat ion was much greater f rom lactate + pyruvate t han f rom prop iona te (Table 1). St imulat ion of gluconeogenesis by palmi- tate was similar in cells f rom lactat ing and control animals (Table 1).

Rates of palmi ta te uptake, esterification, oxidat ion and ketone p roduc t ion were followed in hepatocytes f rom lactat ing and control sheep. There were few differences in palmita te metabol i sm between the two groups, bu t rates of ketone p roduc t ion f rom palmi- tate were significantly higher in hepatocytes f rom lactat ing animals (Table 2). Oxidat ion of and keto- genesis f rom butyra te was similar in b o t h groups (Table 3). In hepatocytes f rom bo th lactat ing and cont ro l sheep, carni t ine significantly s t imulated keto- genesis f rom palmitate , and p rop iona te significantly reduced ketogenesis f rom bo th palmi ta te and bu- tyrate (Tables 2 and 4). P rop iona te had no significant effect on palmita te or butyra te oxidat ion or palmi ta te esterification. Lactate + pyruvate slightly inhibi ted ketogenesis f rom palmita te and butyra te bu t stimu- lated their oxidat ion markedly; esterification of palmita te was also significantly increased (Table 4).

DISCUSSION

Prop iona te is p robab ly the major precursor of glucose in the ruminan t liver (Bergman, 1973; Leng, 1970) but rates of gluconeogenesis f rom prop iona te in isolated ovine hepatocytes were not elevated dur ing l a c t a t i on - - a t ime when glucose tu rnover is increased two- to three-fold (Bergman and Hogue, 1967). How- ever rates of gluconeogenesis f rom lactate + pyruvate were elevated some two-fold in hepatocytes f rom lactat ing sheep. Previous studies on changes in enzyme activities in sheep liver dur ing lactat ion fit with these observat ions, in tha t pyruvate carboxylase is the enzyme in the gluconeogenic pa thway which increases mos t in activity as a result of lactat ion in the sheep (Smith and Walsh, 1982; Mackie and Campbell , 1972) or cow (Ballard et aL, 1968; Baird and Hei tzman, 1970; M e s b a h and Baldwin, 1983). Thus the increases in hepatic gluconeogenesis required to fuel lactat ion in the ruminan t appear to involve adap ta t ion for the increased use of lactate (as pyruvate) as a gluconeogenic precursor. Increased rates of glucose synthesis f rom prop iona te mus t result mainly f rom the increased substra te supply (resulting f rom increased feed intake) to the liver and increases in liver size (Fell et al., 1972). Increased up take of b o t h lactate and p rop iona te by the liver is observed in lactat ing cows (Lomax and Baird, 1983). In con- trast, in the ra t liver, exper imental condi t ions which enhance the rate of gluconeogenesis (e.g. s tarvat ion or ho rmone t rea tment) in vivo produce large changes

Table 2. Palmitate metabolism in hepatocytes from control and lactating sheep

Control Lactating Propionate Carnitine gmol palmitate/g dry wt/2 hr

Palm±rate - + 188 + 15 (5)* 144 ± 15 (5)* uptake - - 134 + 18 (5) 108 _+ 8 (5)

+ + 143 ± 14 (5)** 117 +_ 12 (5)

Palm±rate - + 11.8 ± 1.2 (8) 12.4 + 0.5 (8) oxidation - - 12.1 __. 1.2 (5) 12.2 + 1.4 (5)

+ + 13.4 + 2.1 (8) 13.5 + 0.9 (8)

Palmitate - + 101 ± 7 (5) 75 __. 9 (5) esterification - - 106 ± 8 (5) 80 ± 8 (5)

+ + 98±7(5) 75±11 (5)

Ketogenesis - + 33 5- 4 (8)** 44 ± 5 (8)~** - - 13 5- 4 (8) 18 + 4 (8) + + 15 ± 2 (8)** 24 ± 4 (8)t**

Hepatocytes were incubated with palmitate (1 mM) with or without propionate (3 mM) or carnitine (1 mM). Rates of ll-'4C]palmitate uptake, oxidation and esterification and ketone production were determined as described in Materials and Methods section. Results are means + SE with the number of animals in parentheses. Values for lactating animals significantly different from control are tP < 0.05. Values in the presence of propionate or carnitine significantly different from correspond- ing value in their absence are *P < 0.05, **P < 0.01.

Effect of lactation on gluconeogenesis and ketogenesis 285

Table 3. Butyrate metabolism in hepatocytes from control and lactating sheep

Control Lactating Propionate pmol butyrate/g dry wt/2 hr

Butyrate oxidation - 99 + 12 (5) 114 + 23 (3) + 106-t-20 (5) 98+31 (3)

Ketogenesis - 171 + 17 (8) 182 + 19 (6) + 114_ 12 (8)* 93 + 12 (6)*

Hepatocytes were incubated with butyrate (3 mM) with or without propionate (3 mM). Rates of [l-t4C] butyrate oxidation and ketone production were determined as described in Materials and Methods section. Results are means + SE with the number of animals in parentheses. Values in the presence of propionate significantly different from those without propionate are *P < 0.05.

in enzyme activities observed in vitro (Ashmore and Weber, 1968).

As with oleate in the rat (Ross et al., 1967; Williamson et al., 1969), in ovine hepatocytes hepatic gluconeogenesis from lactate + pyruvate was stimu- lated by palmitate. This stimulation of two to three fold was similar in hepatocytes from control and lactating sheep and may regulate gluconeogenesis during lactation only if hepatic uptake of fatty acids changes. There was also a small but significant stimulation of gluconeogenesis from propionate by palmitate.

Pregnancy and lactation in the ruminant are periods of increased susceptibility to pathological rates of ketogenesis (Baird, 1977; Bergman, 1971; Leng, 1970; Schultz, 1974). Several theories have been put forward to explain the association between high rates of gluconeogenesis and ketogenesis in rumi- nants. These include shortage of oxaloacetate (Krebs, 1966), or CoA (Lomax et al., 1983), reduced rates of fatty acid esterification (Bergrnan, 1971; Baird, 1977) or regulation by the accumulation of methyl malonyl CoA (Wahle et al., 1983a, b). From the data pre- sented here there are few major changes in the way in which hepatocytes from lactating and non-lactat- ing sheep metabolize palmitate. Esterification was slightly but not significantly reduced and ketogenesis increased some 30% in cells from lactating animals. No differences were observed in the rates of ketone production from butyrate or the rates of oxidation of palmitate or butyrate. Thus the susceptibility of ruminants to ketosis during lactation may not be due to long-term changes within the liver cells. In the rat at peak lactation there is a decreased rate of ketogenesis (Whitelaw and Williamson, 1977).

Two factors which did regulate palmitate metab- olism were exogenous carnitine and the presence of gluconeogenic precursors. Added carnitine stimu- lated ketogenesis two- to three-fold but had no effect on oxidation or esterification. As a higher concen-

tration of carnitine is seen in livers from lactating sheep (Costa et al., 1976) this may be a contributing factor in the susceptibility of ruminants to ketosis during lactation. Stimulation of ketone production by added carnitine has been observed previously in hepatocytes obtained from rats (Christiansen et al., 1976) and sheep (Lomax et al., 1983).

The presence of gluconeogenic precursors inhibited ketogenesis from both palmitate and butyrate, propi- onate being more effective than lactate + pyruvate as has been described previously (Lomax et al., 1983). The mechanism of inhibition appeared to differ for the two precursors; whereas the inhibition by lactate + pyruvate was small and accompanied by a stimulation of oxidation of both palmitate and butyrate and of esterification of palmitate, the inhibition of ketogenesis by propionate was greater and was not accompanied by changes in the rates of oxidation or esterification. The effects of propionate described here differ in some respects from those reported previously (Lomax et al., 1983) where inhi- bition of ketogenesis from palmitate and octanoate by propionate was accompanied by a stimulation of oxidation of both and of esterification of palmitate. However, from our data, the mechanism of propi- onate inhibition of ketogenesis did not involve alternative pathways of palmitate or butyrate metab- olism. Its effectiveness with both fatty acids indicates control at the intramitochondrial level, possibly through a metabolite of propionate such as methyl malonyl CoA (Wahle et al., 1983a, b) or by compe- tition for the available CoA (Lomax et al., 1983). Propionate was equally effective in reducing ketogen- esis in cells from both lactating and control sheep, but an increased utilization of propionate for gluco- neogenesis, because of the increased demand for glucose in lactating animals, may result in the decreased availability of propionate or a subsequent metabolite and hence minimize its antiketogenic effect.

Table 4. Effect of gluconeogenic precursors on metabolism of palmitate and butyrate in hepatocytes from non-lactating sheep

Ketone Gluconeogenic Esterification Ox ida t i on Ketogenesis precursor precursor /~mol ketone precursor converted/g dry wt/2 hr Butyrate - - - - 111 _-4- 12 145 _+ 22 Butyrate Propionate - - 103 __ 14 55 +_ 8** Butyrate L-lactate + pyruvate - - 158 + 24* 119 _+ 29* Palmitate - - 84 + 9 11 _+ 2 51 _+ 6 Palmitate Propionate 85 _+ 8 14 + 2 30 _+ 3** Palmitate L-lactate + pyruvate 109 + 7** 19 + 4* 42 + 4* Hepatocytes were incubated with either [l-i4C]butyrate (3 mM) or [1-14C]palmitate (1 mM) + carnitine

(1 mM) alone or with propionate (3mM) or L-lactate (3 mM)+pyruvate (0.3 mM). Rates of esterifieation, oxidation and ketone production were determined as described in Materials and Methods section. Results are means + SE for six animals. Values significantly different from those with either butyrate or palmitate alone are *P < 0.05, **P < 0.01.

286 Ar~-E FAULr, a~I~ and HELEN T. POLLOCK

Overall the large increases in the rate of glucose synthesis seen during lactation in ruminants are not accompanied by major changes within the isolated hepatocyte, other than an increased capacity for gluconeogenesis from lactate. Similarly the capacity for ketogenesis is increased only slightly. Thus it would appear that the main regulators of gluconeoge- nesis and ketogenesis during lactation in the ruminant are substrate supply and the changes in the intracellular concentrations of metabolites.

Acknowledgement--We thank Dr D. Ford for the care of the animals.

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