ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 217, No. 1, August, pp. 244-250, 1982

The Time Course of Glucagon Action on the Utilization of [U-14C]Palmitate by Isolated Hepatocytes

BALBIR SINGH, HARALD OSMUNDSEN, AND BORGAR BORREBAEK’

Institute elf Medical Biochemistry, University of Oslo, P.O. Box 111.2, Blindmn, Oslo 3, Norway

Received September 28, 1981, and in revised form April 2, 1982

The time course of glucagon action on the utilization of [U-‘4C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and COz. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and i4COz, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-14C]palmitate.

Glucagon stimulates the oxidation and inhibits the esterification of fatty acids in the liver (1) and in isolated hepatocytes (2). Evidence has been presented that these effects at least in part may be caused by inhibition of acetyl-CoA carboxylase leading to a lower level of malonyl-CoA. Since malonyl-CoA is an inhibitor of car- nitine acyltransferase I, the result is in- creased formation of acylcarnitine from acyl-CoA and the utilization of fatty acids is directed toward oxidation at the cost of esterification (reviewed by McGarry and Foster (3)). Another possible mechanism is decreased rate of esterification due to the fall in the concentration of glycero- phosphate (sn-glycerol3-phosphate) which is the result of increased rate of gluco- neogenesis (4-6). Such indirect effects on fatty acid metabolism would possibly ap- pear later than the direct effects of the hormone. We, therefore, investigated the

i To whom all correspondence should be addressed.

early time course of glucagon action on hepatocyte palmitate oxidation/esterifi- cation.

It is shown in the present report that the effect of glucagon on the formation of acylglycerols and oxidation products from [U-14C]palmitate was delayed in time as compared with the effect of the hormone on gluconeogenesis. This delay corre- sponded to the establishment of a lower glycerophosphate level. Increased rates of Krebs cycle and gluconeogenesis resulted in increased incorporation of fatty acid carbon into glucose.

MATERIALS AND METHODS

Chemicals [U-W]Palmitate was obtained from the Radiochemical Centre, Amersham, United Kingdom. Essentially fatty acid-free bovine serum albumin (Sigma A-6003), glucagon (Sigma G-4250), enzymes, and biochemicals were purchased from Sigma Chem- ical Company, St. Louis, Missouri. Other reagents were commercially available products of analytical grade.

0003-9861/82/090244-07$02.00/O 244 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

GLUCAGON ACTION ON HEPATOCYTE FATTY ACID METABOLISM 245

Isolation of liver cells. Hepatocytes were isolated from livers of male Wistar rats (200-250 g). Unless otherwise stated, cells from rats which had free ac- cess to a standard pellet diet and water were used. Fasted rats had free access to water, but were de- prived of food 24 h before they were killed. The pa- renchymal liver cells were prepared and purified ac- cording to Seglen (7).

Incubation conditions. Krebs-Henseleit bicarbon- ate buffer containing fatty acid-free bovine serum albumin was used throughout as cell suspension me- dium. Incubations were performed under oxygena- tion (95% Oz and 5% CO.& at 37°C and initiated by mixing equal volumes of cell suspension and medium (containing labeled palmitate). The incubation mix- ture contained [U-‘4C]palmitate at indicated concen- trations, 0.4 mM fatty acid-free serum albumin and 4-7 mg cell protein/ml. Glucagon (dissolved in 1 mM HCl) was added 10 min after the start of incubation to a concentration of 25 nM. At given time intervals, 2-ml samples were taken and mixed with 0.1 ml 70% perchloric acid. When ‘*CO2 was measured, incuba- tions were performed with 2 ml incubation mixture in 25-ml Erlenmeyer flasks stoppered with a rubber cup and provided with a suspended plastic cup sup- plied with folded filter paper. Glucagon and perchlo- ric acid were injected through the rubber stopper. Immediately after the addition of perchloric acid, phenylethylamine/methanol (l:l, v/v) was injected into the suspended cup and the flasks were incubated with shaking for another 60 min. The suspended cups with the collected COz were then transferred to scin- tillation vials with 5 ml of scintillation medium (Packard Instagel II), thoroughly shaken, and counted. Radioactivity was determined with a Packard liquid scintillation spectrophotometer Model 3255.

Enzymatic analysis. The perchloric acid extracts were neutralized with K,COa and used for enzymatic determinations of glycerophosphate (8), lactate (9), glucose (lo), acetoacetate (ll), and P-hydroxybutyr- ate (12).

Deternzinatim of acid-soluble radioactive products. The neutralized perchloric acid extracts were used for determination of acid-soluble radioactivity. Ali- quots (0.2 ml) of the extracts were diluted with 1 ml of water and applied on columns (1.5 X 0.7 cm’) of Bio-Rex 5 anion exchanger (Cl-form). The columns were then washed with 3 X 0.5 ml of water. The ra- dioactivity of the pooled filtrates (the nonanionic fraction) was that of glucose. The radioactivity that was retained on the column (the anionic fraction) was eluted with 2 X 1 ml of 1 M NaCl. This fraction consisted of ketone bodies and metabolic interme- diates. The amount of radioactivity applied on the columns was completely recovered as the sum of total radioactivity of the two fractions.

High-pressure liquid chromatography. Chromatog- raphy of the neutralized perchloric acid extracts were

performed with a Spectra-Physics 8000 chromato- graph using 13 mN HzSOl and an Aminex HPX 81 column connected with a Model BF 5026 Berthold radioactivity detector and a Model 770 Spectra-Phys- ics spectrophotometric detector.

Determination of labeled acylglycerob and fatty ac- ids. The precipitate after centrifugation of the per- chloric acid-treated incubation mixture was ex- tracted with chloroform/methanol and the lipids were separated by thin-layer chromatography (13). The spots were scraped into scintillation medium for counting.

Presentation of the results. The radioactivities of lipids and acid-soluble oxidation products are pre- sented as nanomoles of [UJ4C]palmitate incorpo- rated, i.e., counts per minute (cpm) of the various fractions divided by the specific activity (cpm/nmol) of the palmitate present in the incubation medium. The data represent means of results with four to six different hepatocyte preparations.

The basic rate of palmitate utilization varied some- what from the one cell preparation to the other. However, the described effects in figures and tables were reproducible with each cell preparation. Entire time curves (Fig. 1) were obtained with each prep- aration. Control and glucagon-treated cells were in- cubated simultaneously. In the experiments de- scribed in Tables I and II, paired samples, one with and the other without glucagon, were obtained with each single-cell preparation. The separate differ- ences produced by glucagon in each pair were cal- culated and used for direct determination of the stan- dard error of the difference which was employed in Student’s t test.

RESULTS AND DISCUSSION

The eflect of glucagon cm the formation of labeled acylglycerols and acid-soluble ox- idation products. As is evident from Fig. 1, several minutes after addition of glu- cagon were required before the increased oxidation/esterification ratio could be ob- served. Increased oxidation was largely balanced by decreased esterification, but some enhancement of palmitate utiliza- tion was observed after 10 min of in- cubation in the presence of glucagon (not shown). Initially, glucagon decreased the formation of acid-soluble radioactive products (Fig. 1).

Fractionation of acid-soluble labeled products by high-pressure liquid chroma- tography. In order to investigate the pos- sibility of significant acid-soluble radioac- tive products other than ketone bodies,

246 SINGH, OSMUNDSEN, AND BORREBAEK

foL C 0 IO 20 30 LO

Time (min)

FIG. 1. The effect of glucagon on the incorporation of radioactivity from 0.45 IXIM [U-‘%]palmitate into acid-soluble oxidation products (0,O) and total acyl- glycerols (A, A). Filled symbols indicate the presence of glucagon added after 10 min incubation while open symbols represent controls with no glucagon added. Hepatocytes from fed rats were used.

aliquots of the neutralized perchloric acid extracts prepared after 30 min incubation with 0.15 mM palmitate in the presence of glucagon were analyzed by high-pressure liquid chromatography with on-line radio- activity detection. As is evident from Fig. 2A, two main peaks of radioactivity were observed. Peak 2 apparently represents the radioactivity of the ketone bodies. Fig- ure 2B shows the results when the sample was pretreated with hexokinase and ATP. Most of the radioactivity in peak 1 was transferred to another elution fraction (peak 3). This experiment (Fig. 2B) was performed with a limiting amount of ATP (less ATP than glucose in the extract). With excess ATP, all radioactivity of peak 1 was transferred to peak 3 (Fig. 2C). These results show that the acid-soluble radioactive products could be divided into two main fractions, one consisting of glu- cose (peak 1) and the other mainly of ke- tone bodies (peak 2). The extracts also con- tained labeled metabolic intermediates in amounts too small for detection by the radioactivity detector (some possibly cov- ered by peak 2).

A similar quantitative distribution into two fractions as that shown in Fig. 2A was obtained by filtering the extracts through

anion-exchange material. Glucose was not retained by the anion exchanger while the other fraction, which contained ketone bodies and other anionic compounds, was retained and later eluted with NaCl. Less than 20% of the radioactivity of the an- ionic fraction remained after lyophiliza- tion at pH 3 followed by several times ex-

‘IA 1 1 I 1

0 5 10 15 20 Time of run (mln)

FIG. 2. High-pressure liquid chromatography of the neutralized perchloric acid extract of the sample after 40 min incubation with 0.15 mM m-“Clpalmitate in the presence of glucagon (added after 10 min). (A) Untreated extract (100 pl injected). (B) After treat- ment with 3.5 mre added ATP/M%+ and hexokinase (1 U/ml of yeast hexokinase, Sigma H-5750) for 15 min at room temperature. (C) After same treatment as above, but with 10 mM added ATP/Me. The graphs in A-C show radioactivity detections. The lower curve of A shows the optical density profile (210 nm) together with an indication of the elution patterns of &hydroxybutyrate (BOHB) and aceto- acetate (Ac.ac.) when applied separately to the chro- matography column. Hepatocytes from fed rata were used.

247 GLUCAGON ACTION ON HEPATOCYTE FATTY ACID METABOLISM

TABLE I

THE EFFECT OF GLUCACON ON THE INCORPORATION OF RADIOACTIVITY FROM 0.15 mM [UJ’CIPALMITATE INTO i4C02 AND ACID-SOLUBLE OXIDATION PRODUCTS“

Acid-soluble radioactive oxidation products

Incubation time Nonanionic (min) “CO* Anionic (glucose)

10 Control 0.15 0.88 0.19

12 Control 0.19 0.95 0.22 Glucagon 0.25 0.82 0.27 Difference +0.06 f 0.006* -0.13 + 0.01: +0.05 + 0.01**

40 Control 0.67 1.57 0.45 Glucagon 1.79 2.24 3.02 Difference +1.12 + 0.1* +0.6’7 + 0.12** +2.57 + 0.22*

a The data are given as nmol [U-r4C]palmitate incorporated/mg cell protein. Differences are presented with the SE values. Glucagon was added after 10 min incubation. Cells from fed rats were used.

*P < 0.001. ** P < 0.01.

traction with ether. The ion-exchange method for separation of the two radioac- tive main fractions was preferred because of the higher sensitivity of the scintilla- tion counter.

The eflect of glucagon CYPZ the fn-mation of WO, labeled glucose, and labeled anionic com,pou~~&. The initial delay in formation of acid-soluble radioactive products in the presence of glucagon (Fig. 1) was more pronounced when a lower palmitate con- centration was used as in the experiment described in Table I. Glucagon initially inhibited the formation of anionic radio- activity. This was balanced by a stimu- lated generation of labeled CO2 and glu- cose. At 30 min after hormone addition, glucagon had caused a 6- to ?-fold increase in the flux of radioactivity into glucose and a 2- to 3-fold increase in 14C02. Also an- ionic radioactivity was increased (about 1.5-fold) at this long incubation time (Ta- ble I).

We have so far not been able to identify and determine the radioactivity of the dif- ferent labeled metabolic intermediates which are present in the anionic fraction together with the ketone bodies. Never- theless, it seems reasonable to anticipate that the total acid-soluble radioactivity

plus 14C02 represent the total flux of ra- dioactivity through P-oxidation, and that glucagon initially increases the flux into glucose at the cost of ketogenesis and/or formation of labeled metabolic interme- diates. At prolonged incubation, the flux through ,!?-oxidation was stimulated by the hormone, and all labeled oxidation prod- ucts were increased.

Stimulation by glucagon of the flux of labeled carbon atoms from palmitate into glucose must imply increased flux through the Krebs cycle as well as through B-oxi- dation and gluconeogenesis. This may in- volve stimulatory effects of this hormone on Krebs cycle enzymes and oxidative phosphorylation, which have been re- ported (14). The phosphoenolpyruvate car- boxykinase reaction probably contributes significantly to the 14C0, formed in the presence of glucagon.

The effect of glucagon on enzymatically determined glucose, lactate, and ketone bodies. A considerable amount of endoge- nous lactate was present with cells from fed rats. Most of this lactate was utilized during 30 min incubation in the presence of glucagon, and the hormone effect was evident after 2 min incubation (Table II). The large amount of glucose produced in

SINGH, OSMUNDSEN, AND BORREBAEK

TABLE II

THE EFFECT OF GLUCAGON ON THE LEVELS OF GLUCOSE, LACTATE, AND KEWJNE BODIES“

Incubation time (min)

10

12

40

Control

Control Glucagon Difference

Control Glucagon Difference

Glucose Lactate

190 225

196 231 228 212 +32 t- 8.5 -19 f 4.2**

356 227 960 34

f604 + 17* -193 + 15*

Aceto- acetate

7.65

8.12 7.76

-0.36 iz 0.19

18.4 26.7 +8.3 k 0.8*

@Hydroxy- butyrate

3.59

3.89 4.58

+0.69 + 0.14*+

8.10 6.59

-1.51 + 0.43

“The data are given as nmol/mg cell protein. Differences are presented with the SE values. Giucagon was added after 10 min incubation. The initial concentration of palmitate was 0.15 mhf. Ceils from fed rats were used.

* P < 0.001. ** P < 0.01.

the presence of glucagon was due to acti- vated glycogenolysis.

The amounts of ketone bodies originat- ing from palmitate (anionic fraction, Ta- ble I) were small as compared with the total amounts of ketone bodies (Table II), which were formed also from carbohy- drate sources. This impeded the possibility to test the initial inhibitory effect of glu- cagon on anionic radioactivity (Table I) by enzymatic ketone body determination. Thus, no significant difference in total ke- tone bodies 2 min after hormone addition could be demonstrated. However, there was a significant initial increase in the @-hydroxybutyrate/acetoacetate ratio (Table II).

A rapidly established increased @hy- droxybutyrate/acetoacetate ratio by glu- cagon has been reported by Siess and Wie- land (14), while Christiansen found that this ratio was decreased after 30 min in- cubation (2). The results in Table II are in accordance with both these observa- tions. The initial glucagon-induced in- crease in the fi-hydroxybutyrate/acetoace- tate ratio is consistent with a rapid-onset increased flux through the Krebs cycle. The decrease in the same ratio after pro- longed incubation could possibly be re- lated with accelerated use of energy for gluconeogenesis.

The eflect of glucagon on the glycer& phosphate level. In a previous work in our laboratory (6) it was shown that addition of a small amount of glycerol to isolated hepatocytes led to a rapid transient in- crease in glycerophosphate with a simul- taneous decrease in oxidation and increase in esterification of palmitate. This indi- cated that fatty acid oxidation/esterifi- cation could be regulated by the glycero- phosphate level.

Several minutes of incubation after glu- cagon addition was required before the lower level of glycerophosphate was es- tablished (Fig. 3). A similar time relation for the decrease in malonyl-CoA was ob- served by Cook et al. (15). The delayed ef- fect of glucagon on palmitate oxidation/ esterification (Fig. 1) was, therefore, as expected, if these metabolites are the main regulators. Thus, the results are in accor- dance with an indirect effect of glucagon on fatty acid utilization, the direct effects being stimulated gluconeogenesis and in- hibited lipogenesis. Still, a possible slow action directly on enzymes involved in the oxidation/esterification pathways is not ruled out. Inhibition of hepatic glycero- phosphate acyltransferase by dibuturyl cyclic AMP has been reported (16).

Inwrpwation of curbon atoms from pal- mitate into glucose in hepatowtes from

GLUCAGON ACTION ON HEPATOCYTE FATTY ACID METABOLISM 249

fasted rats in the presence of alanine. A remarkably large part of the carbon atoms from palmitate was transformed to glu- cose in the presence of glucagon (Table I). In this experiment, a relatively low initial concentration of palmitate was employed. With higher palmitate concentrations or with cells from fasted rats, a larger part of the radioactivity was transformed to ketone bodies (data not shown). We were, however, curious about the quantitative significance of incorporation of carbon at- oms from the fatty acid into glucose in a situation which is more comparable to a physiological gluconeogenetic situation, for example, in the fasted state with ala- nine as the gluconeogenetic precursor, and in the presence of a high fatty acid con- centration. Hepatocytes from fasted rats execute a high rate of fatty acid oxidation which is not further stimulated by glu- cagon (2). As shown in Table III, little glu- cose was formed from other sources than the gluconeogenetic precursor (endoge- nous lactate is very low in cell prepara- tions from fasted rats). About 6 nmol of palmitate carbon, equivalent to 6 X 2.7 = 16 nmol of glucose carbon, were incor-

Glucogon

I

I I I 1

IO 20 30 LO

Time (mlnj

FIG. 3. The effect of glucagon on the level of glyc- erophosphate (initial concentration of palmitate was 0.45 mM). Open and filled symbols as in Fig. 1. Bars indicate SE values. Hepatocytes from fed rats were used.

TABLE III

INCORPORATION OF RADIOACTIVITY FROM [U-i%]- PALMITATE INTO ACID-SOLUBLE OXIDATION PROD-

UCTS AND THE FORMATION OF GLUCWE BY HEPATOCYTES FROM FASTED RATS IN

THE PRESENCE OF ALANINE’

Acid-soluble radioactive oxidation

products

Nonanionic Alanine Glucagon Anionic (glucose) Glucose

- + 38.2 2.0 14

+ - 31.6 6.4 142

+ + 30.2 6.8 157

“Cells were incubated for 40 min. Acid-soluble labeled products are presented as nmol [U-r’C]palmitate incorpo- rated/mg cell protein and glucose a8 nmol/mg cell protein. The initial concentrations of palmitate and alanine were 1 and 5 mbr, respectively.

porated into glucose while about 150 nmol of glucose were produced through gluco- neogenesis. Thus, as much as about 10% of the glucose carbon, formed through glu- coneogenesis, originated from the fatty acid. A similar comparison with cells from fed rats is difficult to make since a con- siderable amount of the glucose comes from glycogen breakdown.

We would like to emphasize that the presented results do not counteract the generally held view that fatty acids do not contribute to net glucose synthesis. Each carbon atom incorporated from palmitate into glucose involves one carbon atom from another source (e.g., alanine or lac- tate), expelled as COZ.

ACKNOWLEDGMENTS

This work was supported by grants from the Nordic Insulin Foundation and from the Nansen Foundation. The skilled technical assistance of Mette Ostby is gratefully acknowledged. We would like to thank Dr. Jon Bremer for valuable discussions.

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