Restoration of glycogenesis in hepatocytes from starved rats

Life Sciences Vol . 20, pp . Printed in the U .S .A .

1027-1034, 1977 .

Pergamon Press

RESTORATION OF GLYCOGENESIS IN HEPATOCYTES FROM STARVED BATS Math J .H Geelen Laboratory of Veterinary Bioohemistry, State University of Utrecht, Utrecht, The Netherlands Elizabeth L . Pruden and David M . Gibson Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana 46202 (Received in final form February 10, 1977)

s7]i11RIßTV Hepatooytes isolated from the liver of rate starved for two days synthesized glycogen only when incubated in the presence of both glucose and glucogenio precursors (combinations of alanine, glycerol, pyruvate, lactate or fructose) . Unlabeled gluoogenio precursors facilitated the incorporation of [II- 1 40]glucose into glycogen . Unlabeled glucose likewise greatly enhanced glycogen synthesis from isotopically labeled lactate and other glueogenio precursors . In those systems which contained no added endoorines glucose dampened glycogen phosphorylase activity in a oAMP-independent fashion . Fructose is unable to mimic the effects of gluoose on glycogen deposition and on glycogen phosphorylase activity . The intact rat is too complex for obtaining information on the many sequential changes which take place within the liver in switching from a "gluoogenio" state to a "lipogenio" state . With the advent of improved methods for the isolation of liver cells (1,2), it is possible to examine many metabolic parameters easily and efficiently . Berry,and Friend (1) reported that isolated liver cells from fasted rate are unable to synthesize glycogen, a feature these cells shared with perfused liver (3) . Recently, however, Hems et al . (4) were able to show hormone-independent glycogen accumulation in perfused livers from rate starved for 48 h . It was, therefore, of interest to reinvestigate whether hepatooytee from starved rats are also capable of glycogen deposition . In the present study, an examination of hepatocytes obtained from rats starved for 48 h was undertaken in order to define conditions that permit glycogen synthesis following the provision of metabolic precursors in vitro . The initial evidence suggests that it is possible with these liver cell preparations to accumulate glycogen and to explore the early changes that ensue in the transition from the "gluoogenic" state of liver metabolism in starvation to the "lipogenio" state in refeeding . 1027

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Glycogenesis in Rat Repatocytes

Vol . 20, No . 6, 1977

Methods end materials Male Wistar rate weighing 200-250 f were used throughout these studies . Prior to use the animals were starved for 48 h . The cell isolation prooedure was essentially that described by Ingebreteen and Wagle (2), a modification of the method of Berry and Friend (1) . Two ml of the final cell suspension (150-200 mg wet weight of cells) were added to Krebs bicarbonate buffer with or without substrate providing a final incubation volume gf 3 .0 ml . Incubations were conducted in 25 ml erlenmeyer flasks at 37 for one hour in a Dabnoff metabolic incubator (90 osoillations/min) . Daring incubations vessels were oontinously gassed with 95% oxygen and 596 carbon dioxide . On completion of incubation, flask contents were transferred to iced containers appropriate to the several analyses below and rapidly processed thereafter . 14 Glycogen was isolated by ethanol precipitation . [ C]labeled glycogen was dialysed (5) and an aliquot taken for scintillation counting, employing the fluid described by Patterson and Greene (6) . Glycogen gluoose released following said hydrolysis was determined by the glucose oxidase method (7) . The rate of glycogen synthesis has been corrected for the amount of glycogen present after 60 min of incubation in the absence of added substrates . Glycogen phosphorylase (EC . 2 .4 .1 .1) assays were performed by the method of Robison et al . (8) with the freshly prepared 100,000xg supernatant . CAMP was determined according to the method of Gilman (9) and for this purpose flask contents were treated with triohlorosoetic acid to give a final concentration of 596 . For cell wet weight a 2 .0 ml aliquot was centrifuged in tared tubes for 10 min in the cold at 1,800xg . The supernatant was decanted, the pellet allowed to drain for 30 min in the cold, and the tubes reweighed . The protein content of the cell suspension was determined with biuret reagent, as described for particulate preparations by Cleland and Slater (10) . All incubations and assays were conducted in duplicate . Experimental values reported are the mean of at least four d9jerminations . Collagenase, Type II, was obtained from Worthington, [C]labeled compounds from New England Nuclear ; all other reagents from Mallinckrodt or Sigma . Results The rate of glycogen formation by suspensions of "starved" hepatocytes was influenced by the nature of the substrates in the incubation medium Fig . 1) . The combination of glucose (30 mM) and gluoogenio precursors ~5 mM each of pyruvate, &lenirle and glycerol) led to the synthesis of more glycogen during 60 min of incubation than the sum obtained with either group added separately . Lactate (10 mM) could replace pyruvate plus glycerol in the glucogenic mixture . Regardless of the presence of other gluoogenio precursors, alanine further enhanced glycogen formation whenever it was added to mixtures containing glucose . Fructose (10 mM) was the most potent gluoogenio substrate tested as long as glucose (30 mM) was present in the medium . Fructose (30 mm) could not, however, replace glucose (30 mm) as a component in the standard incubation mixture of hexose plus gluoogenic precursors . In order to measure the contribution of the several components in the formation of glycogen, experiments were performed with [ 14 C]labeled substrates . In the first experiment shown (Table 1) unla~ led glucose etrAingly increased (over 30-fold) the net flux of [II- 1 `FC]slanine or ]lactate into glyoo~en . Unlabeled lactate and alanine facilitated [IIthe incorporation of [II- 1 fC]glucose into glycogen, even though the basal level with glucose alone was relatively high in this experiment as

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Glycogenesis in Bat Hapatocytes

Vol . 20, No . 6, 1977

A

B

C

x x x x x x xi x

x .x : : x x x

x x x x x x x

aoo

1a0 1a0 140 m IM C) m f00

W A

00 40

m atuooa ALAIIDR

PYRUVAT! GLYCZIDL

m. a. a.

mM MM

MM a . MM

mm le. MM PR11ClOai w. r

LAClATC

FRUCTOM

10.

x x

x

x

x

x

x x

x

x

x x x x

Hot glycogen deposition from glucose and gluoogenio precursors by hepatooytes isolated from rate starved for 48 h . All values in experiments A, H and C are expressed as percent of the net glycogen formed with the incubation mixture containing $luoose (30 MM) and alanine, pyruvate and glycerol (5 mm each) . In experiment A the 100% level is 6 .77 Eannoles of glyoogen glucose per g wet weight of oells per h ; in experiment H, 3 .87 ; and in experiment C, 7 .03compared to basal glucose incorporation shown in Tables 2 and 3 . The interdependence of glucose and gluoogenio precursors was also reflected in the quantity of glycogen formed . Since the amount of glycogen which accumulated per hour in the presence of all three substrates ranged between 11 .15 and 11 .65 poles of glycogen glucose in the separate incubations, an estimate of the contribution of each substrate could be made . Alanine-provided approximately 7 percent of the total carbon, lactate 28 percent, and glucose the remaining 65 percent . In the absence of slanine, lactate and gluooee gave rise to all of the carbons for glycogen synthesis, although with this combination the total glycogen produced was less than optimal . In the second experiment (Table 2) unlabeled glucose or alanine, added separately, increased the incorporation of [II- 1 4C]fructose into glycogen five-fold . Added together, they greatly enhanced incorporation of fraotose (fifteen-fold over the fructose baseline) . The effects of alanine are particularly striking since lose than six percent of the total carbon of newly synthesized glycogen is derived from alsnine in the complete system (see line 1 of Table 2) . The influence of glucose and alanine on fructose incorporation was paralleled by the increase in net glycogen formation . Unlabeled fructose or alanine stimulated the incorporation of [II-14C]

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Glycogenesis in Rat Repatooytes

Vol . 20, No . 6, 1977

Table 1 Incorporation of Glucose, Lactate and Alanine During Hot Synthesis of Glycogen Glycogen as Eauoles glucose /g wet wt/h

Substrates

1 14C ]alanine

[14C ]alanine + [14C ]alanine + + lactate [140]lactate [140]lactate + [14C]lactate + + alanine 1 14C]glucose [14C]glucose + [14C]glucose + + alanine

Flux as jimoles substrate inoorporated /g vet wt/h

Percent contribution of labeled substrate

glucose glucose

0 .26 10 .83 11 .15

0 .04 1 .48 1 .76

7 .7 6.8 7 .9

glucose glucose

0 .58 8 .99 11 .65

0 .14 4 .24 6 .76

12 .1 23.4 29 .0

lactate lactate

4.00 6 .32 11 .25

3 .72 5 .24 7 .75

93.0 82.7 68.8

Hepatooytes isolated from rats starved for 48 h were incueither 5 mm l-[II- 4C]alanine bated in media oont ( 1 Wi) or 10 mM 1--[ C]laotate (1 PCi) or 30 mM D-[II- 14C] glucose (1 kCi) . Unlabeled substrates alanine 5 mm, lactate 10 mM and gluoose 30 mm were added as indicated in, the table . The flux of labeled substrates is calculated from the rate of incorporation of isotope into glycogen. glucose into glycogen, and enhanced net glycogen synthesis . In the complete system glycogen formation ranged between 14.06 and 14.98 pmoles of glycogen glucose per hour . Again, the sum of percentages of glycogen carbon derived from each of the three substrates was approximately 100% (94 " 8%r in Table 2) . Nearly 50 percent of the carbon in glycogen was derived from fructose, 6 percent from al-ins, and the remainder from glucose . riment (Table 3) unlabeled glucogenio precursors again In the third e facilitated [U-C]glucose incorporation into glycogen . In the complete system, glucose, alanine and glycerol contributed approximately 50, 8, and 22 percent, respectively, of the glycogen carbon . Pyruvate probably provided the remainder . The effect of added glucose in facilitating the incorporation of [14C] labeled glucogenic precursors into glycogen was achieved even though these precursors lead to the production of glucose in liver during starvation (11) . Home et al . (4) showed already that in perfused liver isolated from 48-h starved rats glycogen synthetase is stimulated by a high glucose oonoentration in the perfusion medium . However, due to high oAMP levels in the starved state glycogen phosphorylase is quite active under this condi tion (12) . If the activity of the latter enzyme is not diminished, glyoogen deposition will not occur . Therefore, we initiated experiments to measure the activity of glycogen phosphorylase under the conditions used

Glycogenesis in Rat Hepatocytes

Vol . 20, No . 6, 1977

103 1

Table 2 Incorporation of Glucose, Fructose and Alanine During Net Synthesis of Glycogen Glycogen as Moles glucose /g fret vt/h

Substrates

1 140]alanine

+ fructose + gluooèe

Flux as pmoles substrate inoorporated /g vet wt/h

Percent contribution of labeled substrate

14 .06

1 .52

5 .4

2 .07

0 .50

22 .7

[ 140]fructose + glucose

9 .99

2 .61

26 .2

+ alanine

6 .39

2 .60

40 .7

+ glucose

14 .98

7 .41

49 .5

[ 14 0]glucose 14 [ 0]glucose + fructose 14 [ 0]glucose + alanine 14 [ 0]glucose + fructose + alanine

1 .68

0 .92

54 .8

8 .81

4 " 44

50 .4

6 .62

4 .49

67 " 9

14 .31

5 .71

39 " 9

1 1401fruotose 1 14C]fruotose 1 14 C]fructose

+ alanine

Incubation media contained either 5 mM 1- II- 14 C~alanine 1 ~Ci or 10 mM D-[II- 1 40]fructose (1 VCiç or 3 mM Du- 4C j]gluoose (1 pOi) . Unlabeled substrates, alanine 5 mM, fructose 10 mN and glucose 30 mN were added as indicated . Calculations and other conditions as in Table 1 . to study glyoogenesio . Investigations in the laboratory of Hers (13) have established that glyoogen phosphorylase becomes less active when incubated in vitro with glucose . Glucose presumably promoted the dephosphorylation of .this interconvertible enzyme . In Table 4 data are presented which show that phosphorylase activity in isolated hepatooytes falls in the course of a one hour incubation in the presence of glucose . Fructose at the same oonoentration did not effect a decrease in phosphorylass activity in one hour . The influence of glucose on phosphorylase activity was reflected in the rate of glycogen formation (Table 4) . Incubation of liver cells in the presence of glucose (30 W for 10 min did not depress the level of endogenous oA1+D? . oAMP levels for hepatocytes from rats starved for 48 h were : prior to incubation, 325 pmoles/g vet weight (2 .47 pmoles/mg protein) ; after 10 min incubation in the absence of glucose, 355 pmoles/g wet weight (2 .69 pmoles/mg protein) ; after 10 min incubation in 30 mM glucose, 370 pmoles/g wet weight (2 .81 pmoles/mg protein) . Discussion The presented results are compatible with the view that the activities of the gluoogenio enzymes are well developed in the starved state (14), and that much of the hepatic glycogen which initially aooumulates in rat liver following refeeding after a period of starvation may be derived

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Vol . 20, No . 6, 1977

Glycogenesis in Rat Hepatocytes Table

3

Incorporation of Glucose, Alanine and Glycerol During Net Synthesis of Glycogen Glycogen as Moles glucose /g wet wt/h

Substrates

Flux as Moles substrate inoorporated /g wet wt/h

Percent contribution of labeled substrate

I 14 C]alanine

0 .14

0 .02

7 .8

1 .15

0 .03

1 .3

[ 14 C]alanine + pyruvate + glycerol + glucose 14C]glycerol + alanine [ + pyruvate + glucose 14C]gluooee [ 14 [ C]gluooee + alanine + pyruvate + glycerol

7 .77

0 .35

7 .8

8 .20

1 .72

22 .2

1 .27

0 .94 3 .25

74 .0

7 .01

14 1 C]alanine + pyruvate + glycerol

49 .8

Incubation media contained either 5 MM 1-[II- 14C]alanine (1 ~Ci) or 5 mM [2- 1 4C]glycerol (1 FtCi) or 30 mM D-[II- 1 4C]gluooee (1 WCi) . Unlabeled substrates, alanine 5 mm, pyruvate 5 mM, glycerol 5 mM and glucose 30 MM were added as indicated . Calculations and other conditions as in Table 1 . Table 4 Effect of Glucose and Fructose on Glycogen Phosphorylase Activity Expt .

Additions

Phosphorylase activity incubation time, min 0 30 60

Glycogen as Moles glucose /g wet wt/h

1

None Glucose

0 .206

0 .233 0 .175

0 .190 0 .096

2

None Glucose

0 .340

0 .353 0 .206

0 .256 0 .164

0 .51 6 .85

3

Glucose Fructose

0 .360

0 .190 0 .330

7 .13 2 .05

-

Hepatooytes isolated from rate starved for 48 h were incubated in media cont aining gluoogenio precursors (alanine, pyruvate and glycerol, 5 mM each) with or without addition of either glucose (30 2M) or fructose (30 mM) . Phosphorylase activity is expressed as Moles Pi released/min/mg protein .

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Glycogeneais in Rat Hepatocytes

103 3

from non-glucose precursors (4, 15-18) . The contribution of the gluoogenio precursors in glycogen synthesis is notinsignifioant . In the presence of added glucose, approximately 30-50 percent of the glycogen carbon was derived from these precursors . In these conditions the summation of incorporation of glucose plus preoursors was essentially 100% indicating that endogenous substrates contribute virtually no carbon for glycogen synthesis during the incubation of isolated liver cells in the presence of added glucose plus precursors . However, when only alenine, lactate, or fructose were present, the glycogen formed could not be accounted for by the incorporation of these precursors . In these conditions small amounts of glycogen are synthesised presumably from endogenous substrates . When using the complete systems, this contribution of endogenous substrates to the over-all amount of glycogen synthesised becomes negligible . It should also be noted in the present study that gluoogenio precursors were not incorporated into glycogen unless glucose itself was supplied . For example (Table 1), gluo2ge effected a fifteen to thirty fold increase in the net flux of [ C]lactate and [14C]alumna into glycogen . The incorporation of [ 1 4C]fruotose was stimulated significantly by gluóose (Table 2) . Although fructose is an effective precursor of glucose and glycogen (19), it could not replace glucose in assisting the incorporation of other gluoogenio precursors . Glucose can divert glucose-6-phosphate from free glucose formation (typical of the starvation state) into net glycogen synthesis by means of the following meohanisme : (a) Glucose inhibits glucose-6-phosphatase (12, 20-22) thereby slowing glucose release . This enzyme may also catalyse glucose-6-phosphate formation if provided with high concentrations of glucose (23-25) . (b) Glucose can diminish glycogen phosphorylase activity both in liver homogenates and in vivo by direotl stimulating the dephosphorylation of this enzyme (13, 23, 26-301 . (c) Subsequent to the effect of glucose on glycogen phosphorylase, glyoogen synthetase becomes activated (13, 28, 30) presumably by dephosphorylation . In the present study glycogen phosphorylase became indeed dampened when cells were incubated with 30 mM glucose (in the absence of added insulin) . Fructose was much less effective . No changes in oAMP oonoentration could be detected following incubation of hepatooytes with glucose . We also noted that gluoogenio precursors stimulated the incorporation of glucose into glycogen . The influence of alanine was most striking in this regard since it contributed less than ten percent of glycogen carbon in the presence of glucose . These metabolites probably prevent net glucose utilization through the glycolytio pathway as a consequence of active gluooneogenesis in starvation-adapted liver cells . Certain of the precursors employed here may also act as positive effeotore for glycogen oynthetase (31) . The present investigation indicates that liver cells isolated from starved rate can be used to advantage in studying the early metabolic adaptations that take place in liver in switching from a starvation (gluoogenio) state to a refed (lipogenio) state . Aolmowledgements We wish to thank Dr . Donald 0 . Allen for assays of oAMP and to express our appreciation to Drs . William $ . Ingebretsen, Jr ., Zafarul H . Beg and P . John Anderson for many productive discussions in the course of this research . We are indebted to Mrs . K .M .S . Geele n for expert technical assistance .

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Glycogenesis in Rat Repatocytes

Vol . 20, No . 6, 1977

This investigation was supported by grants from the U .S .P .H .S . (HL04219-17), the Indiana Heart Association, and the Grace M . Showalter Residuary Trust . References 1 . M .N . Berry and D .S . Friend, J . Cell Biol . Al 506-520 (1969) . 2 . W .R . Ingebreteen, Jr . and S .R . Wagle, Bioohem . Biophys . Res . Commune . A l 403-410 (1972) . 3 . R . Home, B .D . Rose, M .N . Berry and H .A . Krebs, Biochem . J . 101 284-294 (1966) . 4 . D .A . Hems, P .D . Whitton and E .A . Taylor, Bioohem . J . 529-538 (1972) . 5 . W .Z . Hassid and S . Abraham, Methods in Enzymology (S .P . Colowick and N .O . Kaplan, eds .) Vol . III, pp . 37-38, Academic Press, New York (1957) . 6 . M .S . Patterson and R .C . Greene, Anal . Bioohem . .21 854-857 (1965) " 7 . H .U . Bergmeyer and E . Bernt, Methoden der enzymatischen Analyse (H .U . Ber~eyer, ed .) 2 . Auflage, pp . 1173-1179, Verlag Chemie, Weinheim (1970) . 8 . G .A . Robison, R .W . Butcher and E .W . Sutherland, Cyclic AMP, p . 465, Academic Press, New York (1971) . 9 . A .G . Gilman, Proo . Nat . Aoad . Soi . §1 305-312 (1970) . 10 . K .W . Cleland and E .C . Slater, Bioohem . J . 52 547-556 (1953) " 11 . M .C . Sorutton and M .F . Utter, Annu . Rev . Bioohem . 249-302 (1968) . 12 . H .L . Segal, Science 180 25-32 (1973) " 13 . H .G . Here, H . De Wulf, W . Stalmans and G . Van den Berghe, Advan . Enzyme Regul . 8 171-190 (1970) . 14 . H .A . Lardy, D .O . Footer, Y .W . Young, E . Shrago and P .D . Ray, J . Cell Comp . Physiol . 66 39-54 (1965) . 15 . J .M : Olavarria, O .G .R . Godeken, R . Sandruss and M. Flawia, Bioohim . Biophys . Aota ,1§1 185-188 (1968) . 16 . S .L . Jeffcoate and A .J . Moody, Diabetologia 5 . 293-299 (1969) . 17 . N . Zaragoza-Hermans and J .P . Felber, Horm . Metab . Res . 4 25-30 (1972) . 18 . B . Friedmann, E .H . Goodman, Jr . and S . Weinhouse, J . Biol . Chem . 240 3729-3735 (1965)19 . J .H . Eiton and C .R . Park, J . Biol . Chem . 9A-O 955-957 (1965 " 20 . J . Ashmore and G . Weber, Vitamins Hormones jj 91-132 (1959 21 . G .F . Cahill, Jr ., J . Ashmore, A .E . Renold and A .B . Hasting, Amer . J . Med . 96 264-282 (1959) . 22 . B .E . Ryman and W .J . Whelan, Advan . Enzymol . 3A 285-443 (1971) . 23 . R .C . Nordlie, W .J . Arion, T .L . Hanson, J .R . Gilsdorf and R .N . Horne, J . Biol . Chem . LU 1140-1146 (196e) . 24 . R .C . Nordlie and W .J . Arion, J . Biol . Chem . gAO 2155-2164 (1965 . 25 . R .C . Nordlie and D .G . Lygre, J . Biol . Chem . 2e1 3136-3141 (1966 . 26 . P .A . Holmes and T .E . Mansour, Bioohim . Biophys . Acta 156_ 275-284 (1968) . 27 . W . Stalmans, H . De Wulf, B . Lederer and H .G . Here, Bur . J . Bioohem . 9-12 (1970) . 28 . H .G . Hers, H . De Wulf and W . Stalmans, FEBS Lett . 12 73-82 (1970) . 29 . W . Stalmans, H . De Wulf and H .G . Hers, Rum . J . Bioohem . 18 582-587 (1971) . 30 . W . Stalmans, H . De Wulf, L . Hue and H .G . Hers, Arch . Internat . Physiol . Biochim . 8 1 598 (1973).F Kim, J . Biol . Chem . 2A8 2790-2795 (1973) " K 31 . L .N . Magner end .-

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