Biochem. J. (1976) 156, 671-680 Printed in Great Britain

671

Gluconeogenesis in Isolated Intact Lamb Liver Cells EFFECTS OF GLUCAGON AND BUTYRATE By MICHAEL G. CLARK,* OWEN H. FILSELL and IVAN G. JARRETT CSIRO, Division of Human Nutrition, Kintore Avenue, Adelaide, S. Australia 5000, Australia (Received 2 March 1976) 1. Isolated lamb liver cells were prepared from 24h-starved animals by venous perfusion of the excised caudate lobe with buffer containing collagenase. On the basis of Trypan-Blue exclusion, rate of 02 uptake, adenine nucleotide content and retention of constitutive enzymes, these cells werejudged to be intact. 2. Isolated caudate-lobe liver cells showed rates of gluconeogenesis from O0mM-propionate and 10mM-lactate that compared favourably with rates determined in isolated median-lobe cells and with rates determined with the isolated perfused lamb liver. 3. The gluconeogenic potential of substrates tested depended on the lamb's age. Cells prepared from suckling lambs (up to 20 days of age and essentially non-ruminant) showed highest rates from galactose, serine and alanine; those prepared from post-weaned lambs (older than 30 days of age and ruminant) showed highest rates from propionate, lactate and fructose. 4. Gluconeogenic rates from endogenous precursors, 10mM-propionate and I0mM-galactose, were linear for 1 h and were both stimulated by 1 pM-glucagon. Provided the endogenous rate of gluconeogenesis remained unchanged after substrate addition, glucagon caused a net stimulation of gluconeogenesis from each of these substrates. 5. Gluconeogenic capacity and glucagon sensitivity were examined in cells maintained in substrate-free oxygenated buffer at 370, 220 and 0°C. Even under the best of the three conditions of storage that were tested (i.e. at 22°C in gelatin-containing buffer) deterioration of the lamb cells proceeded rapidly, and loss of glucagon responsiveness preceded the loss of ability to convert precursor into glucose. 6. n-Butyric acid, 2-methylpropanoic acid and 3-methylbutanoic acid at concentrations comparable with those found in lamb portal-vein blood each stimulated gluconeogenesis from 10mM-galactose or 10mM-propionate; gluconeogenesis from galactose was stimulated to the greater extent. 7. The regulatory effects of glucagon and sodium butyrate on lamb liver-cell gluconeogenesis and glycogenolysis were compared. Glucagon (1 uM) and 2mM-butyrate accelerated the rate of glucose formation in liver cells of 24h-starved animals from lactate+pyruvate or fructose. Insulin (20nM) decreased both gluconeogenesis and the efficacy of 1 uM-glucagon. For lactate + pyruvate as substrate, the stimulatory effect of butyrate was additive to that of 1 uM-glucagon and for both lactate + pyruvate and fructose the stimulatory effect of butyrate was not influenced by 20nM-insulin. In contrast with glucagon, which stimulated the rate of glycogenolysis in cells prepared from fed lambs, butyrate (0.1-20mM) had no effect. 8. It is concluded that glucagon and butyrate stimulate lamb liver-ell gluconeogenesis by different

mechanisms. The use of viable and morphologically intact isolated liver cells from the rat offers many advantages for studying aspects of liver metabolism, particularly when metabolic properties ofthe cells closelyresemble known properties of the tissue in vivo. The method of choice for the preparation of rat liver cells is that of Berry & Friend (1969), which involves perfusion of the rat liver with collagenase and hyaluronidase. As there is a general paucity of information on hepatic gluconeogenesis and its regulation in larger animals, possibly owing to the cumbersome nature of large * Present address: Clinical Biochemistry Unit, School of Medicine, The Flinders University of South Australia, Bedford Park, S.A. 5042, Australia.

Vol. 156

perfusion, the present work is concerned with the gluconeogenic properties of liver cells prepared from the excised caudate lobe ofthe lamb. The regulation of hepatic glucose formation by glucagon and butyrate is compared. A preliminary communication of some of these results has been published (Jarrett et al., 1975). liver

Materials and Methods Materials

Substrates, enzymes, coenzymes and general reagents were of analytical grade from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and BDH Chemicals

672

M. G. CLARK, 0. H. FILSELL AND I. G. JARRETF

(Poole, Dorset, U.K.). Collagenase was obtained from either Worthington Corp., Freehold, NJ, U.S.A. (type CLS) or Sigma Chemical Co. (type I) and had an activity of approx. 200units/mg (1 unit will liberate amino acids from collagen equivalent to ninhydrin colour of 1.Oumol of L-leucine in 18h at pH7.4 and 37°C). Glucagon (Eli Lilly, Indianapolis, IN, U.S.A.; lot no. 258-D30-138-4) was freshly prepared in 0.9% NaCl (0.1 mg/ml). Insulin (40i.u./ml; Actrapid brand manufactured by Novo Industri A/S, Copenhagen, Denmark and supplied by Evans Medical Australia Pty., Boronia, Vic., Australia) was diluted in iso-osmotic NaCl before use. Lambs and sheep were anaesthetized with Sagatal brand of sodium pentobarbitone (60mg/ml) dissolved in water containing 9% (v/v) ethanol and 20% (v/v) propylene glycol (May and Baker, West Footscray, Vic., Australia). Other materials used were heparin (Allen and Hanburys, Boronia, Vic., Australia), gelatin (Difco Laboratories, Detroit, MI, U.S.A.), bovine serum albumin (essentially fatty acid-free), glucose oxidase (type II), peroxidase (type II) and fatty acids (Sigma Chemical Co.). Short-chain fatty acids (C6 and less) were prepared as neutral aqueous solutions (pH7.4). Other fatty acids were neutralized with NaOH and complexed with bovine serum albumin (60mg of protein/mmol of fatty acid). Animals Merino lambs, ranging in agefrom 1 to 7 weeks, were used; they were born during September, October (spring) or December (summer) from ewes that had been grazing on improved mixed pasture. Where indicated, lambs were removed from their dams and kept in cages without food for 24h before experimentation. Adult Merino sheep (2-3 years of age) were starved for 3 days before experimentation. Preparation of isolated liver cells Lambs and sheep were anaesthetized by intrajugular infusion of sodium pentobarbitone (approx. 25mg/kg body wt.). The liver was exposed by a lateral abdominal incision on the right flank of the animal extending from backbone to linea alva. Unless indicated otherwise, the caudate lobe (approx. 4.5 % by wt. of the total liver) was used for the preparation of cells. After intrajugular infusion of heparin (500i.u./ kg body wt.) the caudate lobe was excised from the liver in situ by a single transverse cut and placed on a polypropylene tray with portal-vein blood vessel uppermost. After quickly weighing the lobe, the tray and lobe were positioned in the perfusion apparatus so that a tapered cannula fitted snugly into the portalvein blood vessel. Venous outflow drained directly into the perfusion system. The average time taken

beginning with the transverse cut across the caudate lobe in situ until the cannulation of the isolated lobe was 20 s. Where indicated, median-lobe liver cells were prepared by similarly sectioning the median lobe followed by perfusion in the manner described above. In these experiments, a section representing no more than 7 % by wt. of the liver was perfused. Cells were prepared essentially as described by Berry & Friend (1969) with the perfusion apparatus and modifications based on those of Zahlten & Stratman (1974). The total volume of perfusion medium [Ca2+-free Krebs improved Ringer I containing pyruvate (Dawson et al., 1969)] was 210ml, of which 50ml was discarded with the blood of the caudate lobe at the commencement of the perfusion. Recirculating perfusion was started and continued for a total of 35min at a flow rate of 40-60ml/min. The perfusion medium was pumped to a reservoir located 20cm above the liver lobe. The pH and P02 of the perfusion medium entering the liver were 7.4±0.2 and 400±SOmmHg respectively, and when the system was continuously gassed (see Zahlten & Stratman, 1974) at 3 litres/min with 02+CO2 (95:5) the pH did not require adjustment. Collagenase (60mg) was introduced in the final 20min of perfusion. After removal from the perfusion apparatus, the liver lobe was gently minced with scissors in 30ml of perfusate and eased through a double layer of Terylene mesh (1 mmx 0.4mm), with the aid of a Teflon pestle and 50ml of freshly prepared (and gassed) perfusion medium containing collagenase (0.037 %, w/v). Undigested material originating from poorly perfused areas of the liver proximal to the cut surface did not pass through the mesh and was discarded. The liver suspension (80ml) representing 80 % by wt. of the lobe was incubated with shaking at 37°C for l5min and gassed with the 02+C02 mixture to retain pH and P02 values at 7.4 and 400mmHg respectively. At the end of the incubation, the cell suspension was filtered through two layers of nylon mesh (0.1 mm diam.), and the cels were separated from debris by centrifuging at 1O0g for 75s. The supernatant was discarded and the cells were washed twice at 220C (centrifuging each time at lOOg for 75s) with 20vol. of gassed Ca2+-free Krebs-Henseleit saline (Dawson et al., 1969), containing 1.5% Y(w/v) gelatin. The washed cells were finally suspended in approx. 20 vol. of the gassed gelatin buffer and kept at 220C until used. Cells from fed lambs were prepared in manner identical with that described above, except that all washing procedures and the final suspension were conducted in gassed Ca2+-free KrebsHenseleit saline at 0°C. All cell suspensions were used within 10min of preparation. The yield of cells was approx. 30 % of the initial weight of the lobe. The dry weight of the liver-cell suspension was determined 1976

GLUCONEOGENESIS IN ISOLATED LAMB HEPATOCYTES

673

from the difference in dry weight between 1 ml of cell suspension and 1 ml of gassed buffer, each dried at 80°C for 17h.

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Gluconeogenic rates Rates of glucose synthesis were determined in stoppered glass vials of 20ml capacity incubated with shaking (80 oscillations/min) at 37°C, with substrate and hormone in a total volume of 1.5 ml of the gelatin buffer. After addition of the cells (approx. 8.7mg dry wt. of liver) each vial was gassed with the O2+CO2 mixture and stoppered for the entire 30min incubation. The reactions were stopped by the addition of 0.5 ml of 6% (w/v) HC104. After thorough mixing, precipitated protein was removed by centrifugation (5000g for 10min), and the supernatant was analysed for glucose content by the glucose oxidase method of Huggett & Nixon (1957) in an autoanalyser [Technicon (Ireland) Ltd., Dublin, Republic of Ireland]. Lamb liver perfusion The portal vein and bile duct were cannulated in situ and the liver was surgically isolated and connected to a perfusion apparatus similar to that used above for the preparation of lamb liver cells. The apparatus was contained within a humidified constant-temperature (37°C) cabinet. The following modifications were incorporated: total capacity of the system was increased to contain

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600ml of perfusion medium; pump rate was increased to 500ml/min; liver support and collecting funnel was increased in size to accommodate a 120-140g liver. Other particulars were as follows: perfusion medium was Krebs-Henseleit saline (Dawson et al., 1969); gas was 02+CO2, (95:5) at 51itres/min; samples for glucose analyses were 2ml of perfusion medium mixed with 4ml of 6%Y (w/v) HCI04 then treated as described above.

Table 2. Ghuoneogenesis from lactate and proplonate in lamb liver preparations

Spring lambs (10 days old) were separated from the parent ewe and placed in cages away from food for 24h before experimentation. Methods for the preparation of liver cells from the median and caudate lobes as well as for the perfusion of the isolated lamb liver are given in the Materials and Methods section. The rate of gluconeogenesis was determined by measurement of glucose formation or by incorporation of 14C-labelled substrate into glucose plus glycogen (Ballard, 1971). Rates determined by the latter method are given in parentheses. Values shown are from different animals. Rate of glucose formation (umol/min per g dry wt. of Substrate Preparation cells) Perfusion None 0.90 Perfusion lOM-Propionate 2.66 (1.43) Perfusion 2.41 (1.13, 1.48) 10mM-Lactate Median-lobe cells None 0.49 Median-lobe cells lOmM-Propionate 2.02 Median-lobe cells 10mM-Lactate 1.86, 1.72 Caudate-lobe cells None 0.42 Caudate-lobe cells lOmM-Propionate 1.94 (2.06) 1.97 Caudate-lobe cells 10mM-Lactate 2.07,2.15, 1.68

Determination of metabolites Glycogen was determined by the method of Carroll et al. (1956), ATP by thatof Lamprecht &Trautschold (1963), ADP and AMP by those of Adam (1963).

Results and Discussion Morphology and properties of isolated lamb liver cells Examination by light microscopy showed intact cells to have discrete rounded membranes, with nuclei and granular cellular contents also readily visible (Plate 1). From preliminary cell preparations it was noted that hormonal responses and maximal pre-hormone rates of gluconeogenesis were only obtained with morphologically intact cells. Although cell popula-

Table 3. Effect of development on the gluconeogenic properties of lamb hepatocytes Caudate-lobe hepatocytes were incubated with substrate (10mM, unless indicated otherwise) and buffer at 37°C, as described in the Materials and Methods section. The number of animals from each age group is as shown. The non-ruminant to ruminant transition occurs between 20 and 30 days of age. Where appropriate, mean values ±S.E.M. are shown. Mean rate of glucose formation

(jmol/min per g dry wt. of cells) Age of lamb (days) Substrate None Propionate Acetate Butyrate 2-Methylpropanoate n-Pentanoate (2mM) Tridecanoate (2mM) Lactate Pyruvate Glutamate Aspartate Glycine Serine Alanine Fructose Galactose Glycerol Dihydroxyacetone No. of animals

10

20

30

50

0.42±0.05 1.96±0.20 0.42±0.05 0.42±0.05

0.64±0.07 2.69±0.30 0.64±0.07 0.64±0.07

0.52 2.96 0.52 0.52

2.37±0.32 2.04±0.30 0.83±0.11 0.94±0.27

3.18 2.28

0.32 3.04 0.32 0.32 0.46 1.27 0.42 2.63 1.81

0.64+0.07 0.73+0.10

0.73±0.28 2.41±0.36 4.32±0.60

0.52 0.52 0.64 3.28 3.52

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1.15±0.15 0.62±0.06 2.19+0.26 2.17±0.21 0.68±0.07 0.51±0.12 0.49±0.06 0.72±0.09 1.20±0.25 1.42±0.32 3.06±0.33

1.60 1.40

0.69+0.06

1.70±0.20 5

0.32 0.32 0.60 4.67 2.94 1.02 1.40 2

1976

The Biochemical Journal, Vol. 156, No. 3

Plate I

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Visible nmicrographs of intact isolated sheep liver cells The cells were prepared from (a) 10-day-old and (b) 3-year-old animals (magnification x800). A non-viable Trypan-Bluestaining cell is indicated by the arrow.

M. G. CLARK, 0. H. FILSELL AND I. G. JARRETT

(Facing p. 674)

675

GLUCONEOGENESIS IN ISOLATED LAMB HEPATOCYTES tions that appeared in poor condition were capable of synthesizing glucose, hormonal responses were not detectable. Thus liver-perfusion and cell-isolation procedures, as described in the Materials and Methods section, were adhered to rigidly. Routine microscopic examination of cell suspensions indicated that the parenchymal cell diameter increased during development, with the change occurring approximately at the transition from the nonruminant to the ruminant state (see, e.g., Plates Ia and lb and Fig. 1). This effect was most pronounced when the number of cells per unit dry wt. of cell suspension was expressed as a function of the lamb's age (Fig. 1). Thus for the 1-week-old animal there were approx. 9 x 101 cells/g dry wt. of cell suspension and these cells had a mean diameter of approx. 15pm. The maximum cell size was attained at approx. 60 days of age with approx. 4.5 x 108 cells/g dry wt. and a mean diameter of approx. 25,um. For comparison, liver cells, prepared from 24h-starved adult rat. guinea pig and mouse, occurred at concentrations of 4.1 x 108, 3.4 x 108 and 3.3 x 108 cells/g dry wt. respectively (M. G. Clark, 0. H. Filsell & I. G. Jarrett, unpublished work). The ratio wet wt./dry wt. for lamb liver did not alter significantly between 7 and 60 days of age and was 3.68±0.13 (15) (mean value ±S.E.M.) As shown in Table 1 more than 90% of the cells excluded Trypan Blue and less than 10% of their total contents of lactate dehydrogenase and aldolase were found in the suspending medium. Since fresh lamb liver contained 431 ± 60 (7) units of lactate dehydrogenase per g dry wt. (mean value ±S.E.M.), a loss of approx. 19% of the enzyme occurred during cell preparation. When these cells were incubated for 1 h at 37°C in oxygenated buffer containing utilizable substrate both cell damage and loss of constitutive enzymes increased. Both ofthese characteristics of cell deterioration increased further when either gelatin or substrates were omitted from the suspending medium. The rate of 02 uptake of freshly prepared cells was also determined (Table 1). A basal rate of 8.17pmol/ min per g dry wt. compared favourably with that determined under similar conditions for isolated rat hepatocytes (Berry & Werner, 1974; Lund et al., 1975). Although the addition of 10mM-lactate stimulated the basal rate of 02 uptake for each cell preparation, the assessment of the stimulation for the combined data (three animals) was less than that reported for rat hepatocytes under similar conditions (Berry & Werner, 1974). The adenine nucleotide content of freshly prepared lamb hepatocytes is shown in Table 1. These values compare favourably with similar determinations conducted on freeze-clamped liver taken from the same animals as the cells had been taken from [e.g. mean values ±S.E.M. (three animals) for ATP, ADP and AMP were 10.82±1.21, 3.41 ±0.52andO.87+0.27 pmol per g dry wt. respectively]. Vol. 156

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Fig. 2. Gluconeogenesis from galactose and propionate by isolated lamb hepatocytes The incubation conditions for caudate-lobe hepatocytes were as described in the Materials and Methods section. The incubations were: broken line, control with no additions (U), control+ 1 pM-glucagon (Oi); solid line, 10mMgalactose (A), lO mM-galactose + 1 ,uM-glucagon (A), l 0mmpropionate (0), lOmM-propionate+luM-glucagon (o). The bars are used to indicate the mean values ±S.E.M. for six 10-day-old animals.

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[Glucagon] (M) Fig. 3. Effect ofglucagon concentration on gluconeogenesis from galactose and propionate Isolated lamb liver cells from the caudate lobe were prepared from 24h-starved non-ruminant lambs, as described in the Materials and Methods section. Incubation conditions were as described in the text and additions were: none (U), 2.5 mm-galactose (-) and 5mM-propionate (o).

M. G. CLARK, 0. H. FILSELL AND I. G. JARRETT

676

gluconeogenesis from either lOmM-propionate or 10mM-lactate were similar for all three preparations, whether determined from net glucose formation or from incorporation of radioactive precursor into glucose and glycogen.

Glucose-forming capacity oflamb liver cells Preliminary studies indicated that starving the lambs for 24h before experimentation decreased the liver-cell glycogen content to a value that did not greatly interfere with measurements of gluconeogenesis. Further, although the glycogen concentration was diminished with starvation for lambs of all ages used, the major glycogen loss occurred during preparation of the cells. The mean value for lambs aged 10-20 days, starved for 24h before perfusion, was 55.1±25.9 (4) mg of glycogen/g dry wt. and that for a portion of the digested liver after perfusion was 24.7± 7.3 (4)mg of glycogen/g dry wt. For lambs aged 30-50 days and starved for 24h, the corresponding values were 55.0±8.7 (6) and 36.1±5.9 (6)mg of glycogen/g dry wt. respectively. Gluconeogenic properties of isolated caudate-lobe cells were compared with those prepared from the median lobe and with gluconeogenic properties of the isolated perfused lamb liver (Table 2). Rates of

Effect of development on the gluconeogenic properties of lamb liver cells Seventeen substrates were tested to assess gluconeogenic properties of lamb liver cells prepared from animals ranging in age from 10 to 50 days (Table 3), representing the transition from the non-ruminant to the ruminant state. Of the short-chain fatty acids, propionate was the best substrate and the rate of glucose synthesis increased with the animal's age. No net glucose formation occurred from either butyrate or acetate, but 2-methylpropanoate (isobutyrate) and n-pentanoate were gluconeogenic substrates. Leng (1970) reported that about 19g of glucose/day may

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Time (h) Fig. 4. Effect ofaging ofisolated lamb hepatocytes on the gluconeogenic capacity and glucagon sensitivity Isolated lamb hepatocytes (approx. 16mg dry wt. of cells/ml) were incubated at 22°C (a) and 37'C (b) in Krebs-Henseleit saline buffer, containing 1.5%y (w/v) gelatin, and gassed continuously with the 02+CO2 mixture. The cell suspension was sampled (O.5ml) at the times shown, and 30min incubations were conducted at 37°C to assess gluconeogenic capacity [lOmM-propionate (M)] and sensitivity to glucagon [lOmM-propionate+1 uM-glucagon (0)]. Control rates from incubations of cells alone and cells+glucagon have been subtracted to give net rates of glucose formation. Representative results from two cell preparations are shown. 1976

677

GLUCONEOGENESIS IN ISOLATED LAMB HEPATOCYTES

demand for glucose, associated with the greater glucose-utilization rate of young lambs (Jarrett et al., 1964). Fructose was the best gluconeogenic substrate and this became most evident with development. Rates of gluconeogenesis from galactose were higher than 2.5/umol/min per g dry wt. at all ages and a maximal rate of 3.08,umol/min occurred for 20-dayold lambs. Dihydroxyacetone and glycerol were less effective substrates than fructose.

arise from 2-methylpropanoate and n-pentanoate, derived from ruminal fermentation in the adult sheep. Our data indicate that lamb liver cells have the potential for high rates of gluconeogenesis (equivalent to 30g/day from these two glucogenic short-chain fatty acids). The medium-chain fatty acid tridecanoate also gave rise to some glucose synthesis. Whereas the gluconeogenic rate from lactate increased commensurate with increasing rumen activity, the rate from pyruvate remained constant. Of the amino acids that were tested (Table 3), aspartate and glutamate gave the highest rates of glucose formation, and this occurred in cells from lambs with a developed rumen. For lambs in which the rumen has developed (about 50 days of age) the rates of gluconeogenesis with alanine are about half the rates determined with alanine in adult sheep in vivo by Wolff & Bergman (1972), i.e. about 0.188umol/min per g wet wt. of liver, which is equivalent to an uptake rate of alanine by liver of 3.2mmol/h. However, the rates for gluconeogenesis from alanine and serine were considerably higher for the young non-ruminating lambs than for the weaned lambs. These higher rates may reflect the increased

Stimulation ofgluconeogenesis by glucagon As shown in Fig. 2, gluconeogenicrates from 10mMgalactose and IOmM-propionate were linear for 1 h under the routine incubation conditions, and each rate was stimulated by 1,uM-glucagon. Further, the lamb liver cells were sensitive to glucagon at concentrations as low as 1 nm for 2.5mM-galactose and SmM-propionate as substrates (Fig. 3). To assess the relative lability of gluconeogenic capacity and hormone sensitivity and to assess the relative advantages of differing storage conditions, lamb liver-cell suspensions were maintained under

Table 4. Effect offatty acids on gluconeogenesis in isolated lamb hepatocytes The rates of glucose formation were determined on caudate-lobe cells prepared from non-ruminant spring and summer lambs, as described in the text. The results shown are the means from duplicate determinations, with the numbers of animals given in parentheses. Standard errors have been calculated where appropriate. Rate of glucose formation (pmol/min per g dry wt.) Substrate

Season during which lambs were born ... Additions (2mM) Control None +Substrate Control Acetate +Substrate Control Butyrate

2-Methylpropanoate n-Hexanoate n-Pentanoate 3-Methylbutanoate Octanoate Tridecanoate

Oleate

Vol. 156

10mM-Propionate

lOmM-Galactose

...

Spring

Summer

Spring

Summer

0.42±0.05 (9) 3.06±0.33 (5) 0.42 (2) 3.79 (2) 0.42±0.05 (9) 7.43±0.61 (9) 0.54 (2) 8.63 (2) 0.43 (2) 8.08 (2)

0.51±0.06 (4) 3.03±0.34 (4) 0.51 (2) 3.12 (2) 0.51±0.06 (4) 3.67±0.35 (4) 0.61 (2) 3.30 (2) 0.50 (2) 3.24 (2) 1.15 (2) 3.03 (2) 0.63 (2) 3.21 (2) 0.53 (2) (2) 3.15 0.75 (2) 3.03 (2) 0.60 (2) 3.15 (2)

0.42±0.05 (9) 1.96±0.20 (5) 0.42 (2) 2.14 (2) 0.42±0.05 (9) 2.57±0.12 (4) 0.54 (2) 2.49 (2)

0.51+0.06 (4) 2.20+0.19 (4) 0.51 (2) 2.20 (2) 0.51+0.06 (4) 2.75±0.11 (4)

+Substrate Control +Substrate Control +Substrate Control +Substrate Control +Substrate

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678

M. G. CLARK, 0. H. FILSELL AND I. G. JARREIT

three conditions in substrate-free oxygenated buffer. The experiments of Fig. 4 show that the gluconeogenic capacity for lOmM-propionate decreased by 33% over 4.5h at 22°C, and over the same interval total loss of glucagon sensitivity occurred. An examination of the cell suspension at 4.5 h showed that the number of cells that stained with Trypan Blue was approx. 50%. In addition, many of the cells were clumped together, and at least 20 % of the viable cells had transparent extrusions associated with the membranes. Deterioration of cellular integrity was markedly increased if the cells were maintained at 37°C. Indeed, after only 1 h the gluconeogenic capacity had decreased by 78 % and glucagon sensitivity was not detectable (Fig. 4). Alternatively, if the cells were maintained in gelatin-free and Ca2+-free Krebs-Henseleit buffer at 0°C (results not shown) gluconeogenic capacity decreased linearly to 10% at 4.5h and glucagon sensitivity was totally lost within the first hour. Thus to ensure minimal loss of hormonal responsiveness and gluconeogenic capacity, cells were used immediately after preparation. Effect offatty acids on gluconeogenesis Previous reports that butyrate elevated blood glucose concentrations in the hypoglycaemic lamb (Potter, 1952) and stimulated glucose production by sheep liver (Phillips et al., 1965) provided the impetus to examine the effect of butyrate and other fatty acids on gluconeogenesis in isolated lamb liver cells. As shown in Table 4, 2mM-butyrate, 2mM-2-methylpropanoate, 2mM-2-methylbutanoate and 2mMoleate each accelerated gluconeogenesis from glactose or propionate. This stimulatory effect was most pronounced with galactose in liver cells prepared from lambs born in spring. Of these acids, butyrate and 2-methylpropanoate were the most effective. An examination of the effect of butyrate concentration on gluconeogenesis from lOmM-galactose in spring lambs indicated that concentrations as low as 10puM produced an acceleration in rate (Fig. 5). In contrast with the stimulatory effect of butyrate on gluconeogenesis, no stimulation of glycogenolysis by butyrate occurred in cells from fed lambs. In addition, the failure of butyrate to stimulate glycogenolysis in cells from fed lambs contrasted with the effect of 1 pMglucagon on this rate, which was increased from 8.9±1.2 (5) to 24.9 ±4.5 (5)pmol of glucose released/ min per g dry wt. (mean values ±S.E.M.). The stimulatory effect of butyrate on gluconeogenesis reported in Table 4 was concluded to be of possible physiological significance, as analysis of portal-vein blood from lambs of comparable age indicated the concentration of butyrate to be approx. 0.05mM. The total concentration of butyrate plus 2methylpropanoate plus 3-methylbutanoate was

approx. 0.09 mm, thus being in excess of the minimum concentration of short-chain fatty acid noted to accelerate gluconeogenesis (Fig. 5). Seasonal differences in response to butyrate may relate to different dietary backgrounds. Comparative effects of glucagon and butyrate on gluconeogenesis Table 5 shows that both glucagon and butyrate accelerate the net rate of glucose formation from either lactate+pyruvate or fructose in liver cells from non-ruminant lambs. For lactate+pyruvate the stimulatory effect produced by 2mM-butyrate appeared to be additive to that produced by a supramaximal concentration of glucagon (see, e.g., Fig. 3). Insulin (20nM) decreased gluconeogenesis from each of the substrates (Table 5) and counteracted the stimulatory effect of glucagon on gluconeogenesis.

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[Butyrate] (mM) Fig. 5. Effect ofbutyrate concentration on gluconeogenests and glycogenolysis Isolated lamb liver cells were prepared from the caudate lobe of four spring lambs starved for 24h and from four fed spring lambs, all approx. 10 days of age. Cells from starved animals were incubated with various concentrations of sodium butyrate, pH7.4 (o), + lOmM-galactose (-). Cells from fed animals were incubated with butyrate alone (-). Incubation conditions were as described in the Materials and Methods section. Individual points represent the mean of four separate experiments with the bars indicating the S.E.M.

1976

GLUCONEOGENESIS IN ISOLATED LAMB HEPATOCYTES

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However,- insulin had no effect on the stimulation of gluconeogenesis by 2mM-butyrate. It is noteworthy that glucagon stimulated both the endogenous rate of gluconeogenesis and that from fructose, galactose, lactate+pyruvate and propionate (Table 5 and Figs. 2 and 3). Whereas it is possible that the endogenous gluconeogenic precursors enter the gluconeogenic pathway before the point at which glucagon exerts its stimulatory effect, the existing data (e.g. Table 5) suggest that endogenous gluconeogenesis is attributable to a residual glycogenolysis [glycogenolysis is stimulated by glucagon and also unaffected by butyrate (Fig. 5)]. Assuming that this endogenous rate is not altered by substrate addition, then for each of the substrates listed above, glucagon has caused a net stimulation of gluconeogenesis. Since propionate, fructose and galactose enter the gluconeogenic pathway after pyruvate carboxylase the finding that glucagon stimulates gluconeogenesis from these substrates may support the proposal for a cytosolic site for this agent (e.g. at phosphofructokinase-fructose 1,6-diphosphatase, as proposed previously for rat liver cells by Clark et al., 1974). The stimulation of glucose formation in isolated lamb hepatocytes by physiological concentrations of short-chain fatty acids implies a regulatory mechanism for hepatic gluconeogenesis. The observation that the rate of glucose formation is accelerated from substrates that enter the gluconeogenic pathway after pyruvate carboxylase suggests that the regulation may not operate exclusively at this enzyme.

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Adam, H. (1963) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 573-577, Academic Press, New York and London Ballard, F. J. (1971) Biochem. J. 121, 169-178 Berry, M. N. & Friend, D. S. (1969) J. Cell Biol. 43, 506520 Berry, M. N. & Werner, H. V. (1974) in Alfred Benzon Symposium VI, pp. 751-759, Munksgaard, Copenhagen Carroll, N. V., Longley, R. W. & Roe, J. H. (1956)J. Biol. Chem. 220, 583-593 Clark, M. G., Kneer, N. M., Bosch, A. L. & Lardy, H. A. (1974) J. Biol. Chem. 249, 5695-5703 Dawson, R. M. C., Elliott, D. C., Elliott, W. H. & Jones, K. M. (1969) Data for Biochemical Research, p. 507, Clarendon Press, Oxford Hems, R., Lund, P. & Krebs, H. A. (1975) Biochem. J. 150,47-50 Huggett, A. St. G. & Nixon, D. A. (1957) Lancet ii, 368370 Jarrett, I. G.,Jones, G. B. & Potter, B. J. (1964) Biochem. J. 90, 189-194

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M. G. CLARK, 0. H. FILSELL AND I. G. JARRElT

Jarrett, I. G., Filsell, 0. H. & Clark, M. G. (1975) Proc. Aust. Biochem. Soc. 8, 54 Lamprecht, W. & Trautschold, I. (1963) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.,), pp. 543551, Academic Press, New York and London Leng, R. A. (1970) Adv. Vet. Sci. 14, 209-260 Lund, P., Cornell, N. W. & Krebs, H. A. (1975) Biochem. J. 152, 593-599

Phillips, R. W., Black, A. L. & Moller, F. (1965)Life Sci. 4, 521-525 Potter, B. J. (1952) Nature (London) 170, 541 Wolff, J. E. & Bergman, E. N. (1972) Am. J. Physiol. 223, 455-460 Zahlten, R. N. & Stratman, F. W. (1974) Arch. Biochem. Biophys. 163, 600-608

1976

Gluconeogenesis in isolated intact lamb liver cells. Effects of glucagon and butyrate.

Biochem. J. (1976) 156, 671-680 Printed in Great Britain 671 Gluconeogenesis in Isolated Intact Lamb Liver Cells EFFECTS OF GLUCAGON AND BUTYRATE By...
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