Studies in vitro on single beating rat-heart cells

BIOCHIMICA

E’i

,4TSUKO EUJIMOTQ Labovato~~y Dq5avtment

of Biophysics

AC-IA

AND IS_4AC Il_kR%RY

ofNuclca~ Medicine andRadiatiovc

and Nuclear

School of Medicine,

RIOPNYSICA

Mediciwe,

Center foot the Health Los Aqeles, (Received

Biology,

and Department Sciemxs,

ofBiological Chemists;,‘,

U:nivevsity

ofCul$wnia,

Culil(U.S.A.) _kUgr?st 2nd,

1963)

SUMMARY I.

0,

uptake

by rat-heart

cells decreases

with time in culture.

2. Primary cells have a respiratory quotient of 0.81, which indicates mainly fat metabolism, while cultured ceils have a higher respiratory quotient of 0.96, indicating a shift from fat to carbohydrate metabolism. 3. Both Crabtree and Pasteur effects are established after I week in cuiture, indicating development of a control mechanism centered about glucose metabolism in culture. 4. Glucose and palmitate stimulate 0, uptake of primary cells but lose their stimulatory abilities when the Crabtree effect appears. Pyruvate stimulates 0, uptake of both primary and cultured cells to the same extent. 5. Amytal, at a concentration of I m&I, inhibits 0, uptake of primary cells and this inhibition

increases

with time in culture.

In primary

cells increased

inhibition

of

0, uptake by Amytal is obtained in the presence of glucose, despite the fact that addition of glucose to non-inhibited ceils increases 0, uptake, while addition of palmitate to Amytal-inhibited cells increases Q, uptake. 6. A possible shift from fat to carbohydrate metabolism in cultured rat-heart cells has been discussed.

ISTRODUCTION

Rat-heart cells grown in culture lose their function in from z to 6 week+. Prior to this, enzyme changes occur which may be related to this dedifferentiation or loss of specific function2. Our purpose is to investigate the cause of dedifferentiation in order to obtain information of the factors which control expression of specific function and are related to the presence or absence of specific proteins. These changes are most probably the result of an insufficient nutrition and an improper environment. The requirements for mammalian cells have been worked out in general terms for maintenance of many cell types established in culture, and are Biochim.

Biophys.

Acta,

86 (1964) 7+80

METABOLIC

largely

based upon the ability

maintenance

of function,

SHIFTS

to support

IN CULTURED

CELLS

75

growth 3, 4. The nutritional

however, is still an unexplored

requirement

for

area. Cells grown in a medium

unable to support function may respond by changes in metabolism which serve to adjust to the new environment and permit survival, at the expense of the synthesis of specific proteins. The first step in this process may be a short period of metabolism of endogenous material during which time enzyme changes may occur. We have some indication that this process is occurring in the heart cell. Cultures of heart cells will take up 0, and continue to beat for up to 36 h without any added carbon source. The cells are undoubtedly metabolizing stored material and possibly utilizing structural and functional lipids and proteins. In the presence of glucose in the medium both endogenous

metabolism

be occurring. We have thus initiated

and an adaption

to an altered metabolism

a more general study of the metabolism

in culture to gather information

about overall metabolic

METHODS

may

of the heart

cell

changes that may occur.

AND MATERIALS

The cells derived from 1-3 days old rat hearts were grown in the same manner as described previouslyl. The cells were harvested by trypsinization with 0.15 y. trypsin (EC 3.4.4.4) in the presence of the heart-cell medium minus serum for IO min at 37“. The cells were scraped gently off the plates and collected by centrifugation after being cooled in an ice bath for 5 min. The precipitated cells were washed twice with the isotonic buffer (pH 7.7) described by LEHNINGER~ and suspended in the same buffer for the respiration

studies.

After

uniform

suspension

the cells were placed in 5-ml

Warburg flasks containing 0.05 ml of 20 y0 KOH solution in the center well and 0.1 ml of the appropriate substrate solution in the isotonic buffer in the side-arms. After a 5-min preincubation period the substrates were tipped into the main vessels and measurements

of 0,

uptake

taken

every

IO min. The incubation

was carried

out

at 37’. The respiratory quotient (R. Q.) was determined by measuring the 0, uptake and CO, production in r5-ml Dixon-Keilin flasks which contained 2.7 ml of the cell suspension in the main compartment and 0.2 ml of 5 N H&O, in the side-arms. In certain flasks glucose solution in the isotonic buffer was also added in the main vessels to make its final concentration 0.02 M. After a ro-min preincubation period at 37’, the acid in the flasks for the zero-time controls was tipped in, while experimental flasks were incubated for another 60 min before, the reaction was stopped by the addition of the acids. The 0, uptake and CO, production was calculated by subtracting the amount of CO, in the gas phase and total CO, at zero time from the final experimental readings, respectively. For demonstration of the Pasteur effect the amount of glucose and lactate were determined in the beginning and at the end of the reaction. After a 6o-min incubation period at 37”, the experimental flasks were placed immediately in an ice bath and the reaction mixtures were quantitatively transferred into centrifuge tubes containing 2.0 ml of cold 5 0/0perchloric acid. The denatured protein was precipitated by centrifugation and washed twice with water. The supernatants from the three fractions were pooled and the pH adjusted to 7.5 by the addition of I N KOH. The amount of glucose Biochim.

Biofihys.

Acta,

86 (1964)

74-80

in an aliquo-t of the supesnatant solution was measured using glucose oxidase (p-3glucose: 0, oxidoreductase, EC 1.1.3~4) (glucostat method of VJorthington). Lactate was determined by the method described by OLSOFF. The dry weight of cells was determined by drying them to constant weight. hmytal sodium (sodium amobarbital) was p.urchased from Eli Lilly and CC’. Palmitic acid and lactate dehydrogenase (L-lactate: KAD oxidoreductase, EC i.i.i.27j were obtained grade.

from Cal. Biocbem.

Corporation.

RESULTS ASD Changes

in, 0,

uptaka and respiratory

Ail reagents used were of analytical

DISCUSSION

quotient (R. 0.)

In Table I it is shown that the respiratory activity of the cells decreased with time in culture and fell to about 50 YJ,of the initial activity within 2 weeks. This drop in 0, uptake in cultured cells may be attributed to the decreased ability of the heart celis to oxidize substrate through the tricarboxylic acid cycle, since specific activities of both malate and isocitrate dehydrogenase (L-malate : NAD oxidoreductase, EC 1.1~1.37 and L,-isocitrate : NAD oxidoreductase (decarboxylating), EC I .P, I .41) has been shown to decrease rapidly during the culture period2. The rate of decrease in respiratory activity of the cells, however, was much less than that of the specific activities of these two dehydrogenases. It is possible that these two enzymes are present in great excess in the beginning, and, that the sharp drop observed in z-3 days in cultnre may not represent an equal decrease in 0, uptake.

CHANGES IK RESPIRATORY ~UOTIEKT (R.Q.) Concentration

0

18.6

7

IO.3

14

6.9

26.1 8.2

6.3

40 -20

-9

AND 0,

UPTAKE

of glucose added = o.02 M

1j.I 9.0

6.6

24.2 7.8

6.3

0.81

0.87 0.96

0,93

6.95 1.00

The R. Q. of the cells at various times in culture were determined in the absence or presence of glucose. The final concentration of glucose was 0.02 M. At zero time a value of 0.81 was obtained for the R. Q~ indicating that fat metabolism was the main energy source of uncultured cells. After 2 weeks in culture this value changed gradually to a R. Q. of 0.96, indicating active participation of carbohydrate metabolism in the system. Furthermore, in the beginning an addition of glucose shifted the R. Q. from 0.81 to 0.93, while after 14 days in culture, glucose addition shifted the R. Q~ from 0.96 to 1.0, indicating that a shift of the metabolism from fat to carbohydrate caused by the added glucose is much larger in uncultured cells but decreases with time. The z-week-old cells may already have carbohydrate metabolism as a main source of energy and the additional amount of glucose would not have a significant effect. Many investigators have presented evidence of the intimate role of fat oxidation in heart function. It has been noted, for instance, that fats are actively taken up by the Biochim.

Siophys.

Acta, 86 (1964) 74+h

METABOLIC

SHIFTS

IN CULTURED

77

CELLS

myocardium 7- g, that heart tissue actively metabolizes fat and that the normal heart R. Q. hovers around 0.75 (ref. IO). Crabtree asd Pasteur effect

It can be seen in Tables I and II that the effect of glucose, though maintaining its ability to shift metabolism toward a higher R. Q., changed with time. In the beginning glucose was markedly stimulatory, but as early as the 3rd day in culture this apparent stimulatory effect of glucose disappeared. After I week glucose became an inhibitor and a Crabtree effect was established]-l. It is interesting to note in Table II, that while the glucose effect changed with time, pyruvate remained an active stimulator of 0, uptake. It should be mentioned, however, that although pyruvate stimulated 0, uptake to the same extent through a z-week culture period, the concentrations of enzymes for oxidation of pyruvate appeared to be lowered to about 50 % at the 14th day in culture as the endogenous oxidation fell to about 50 %. TABLE EFFECT

Concentration

OF

GLUCOSE

AND

of additions:

II PYRUVATE

ON

Endogenous

: '4 :

15.3 9.7 7.4 II.8 9.6

14

8.5

0,

UPTAKE

glucose, 0.02 M; pyruvate,

0.002 M.

witI

17.4 8.8 6.0 14.6 7.8 6.7

glucose

20.8 I2.0

-914

IO.0 -

-19 --I9 24 -21

With _+yruvate

36 24 35 -

If we assume that the rate of oxidation of pyruvate to CoASAc remains constant, then the 50 y0 decrease in oxidation of pyruvate, with time in culture, would indicate that the capacity of the primary cells to oxidize CoASAc was twice that of the cultured cells. It would also suggest that the capacity of primary cells to oxidize CoASAc is much higher than that for its formation from pyruvate since pyruvate has been shown to stimulate 0, uptake of both primary cells and cultured cells. Most of the CoASAc in the primary cells would therefore arise from another source, most probably from fat metabolism. This is supported by the fact that palmitate stimulates 0, uptake in primary culture but not in cultured cells (see Table IV). One could therefore speculate that the rate of oxidation inythe tricarboxylic acid cycle in the cultured cells more nearly approximates the rate of oxidation of CoASAc derived from pyruvate, and that the loss in 0, uptake reflects the loss in CoASAc formation from fat. In any case it is quite apparent that two processes are occurring; one, the decreased oxidation capacity of the tricarboxylic acid cycle and two, a lowered endogenous formation of CoASAc. The presence of a Crabtree effect might be an indication of a control mechanism centered about glucose metabolism. The same may be true with the Pasteur effect, the inhibition of glucose uptake by 0, (ref. 12). The results presented in Table III indicate that the uncultured cells do not have a Pasteur effect but develop one with age in B&him.

Biophys.

Acta,

86 (1964)

74-80

culture, measured in terms of gl-ucose uptake and lactate formation. Both the G&tree and Pasteur effects appear to be established at the same time, about 3-4 d2ys 11: culture. It is tempting to speculat, 0 that with the shift from fat to carbohydrate metabolism,

controls

concerned

with

carbohydrate

utilization

are brought

int:;

operation TABLE DEVELO~~~T Concentration

OF PASTEUR EFFECT of glucose Glucose

0

3 6 IO =7 0 ; I3 =7

III

22.3 3.8

@take

added,

0.01 M.

(pmoles/hfier

mg)

Lactate

0.9 0.58 0.64

formed

(,umoZes;h lipr “g,

4.6 4.’

o-93 0.48 0.50 0.65

0.8j

5-8

0.44

0.78

0.7” I.0

14.6 10.8

18.4

0.28

11.5

0.20 0.67

0.62 0.84

12.7

9.8

l.Oj

7.9

i.II

I.j8

9.7

7.3

0.94 0.79 0.78

1.04

10.0

0.44 0.58 0.58

0.81

I.49

22

4 5.2 5.3 8.8

0.47

I.Oj

1.3”

o.ilg

0.94 a.91 1.72 :.qh

0.85

0.46 1.j'

-

Effect of Amytal 01/z0, q%ake The effect of amytal on 0, uptake has been investigated in the attempt to study alterations in mitochondrial function during the cell culture period. Table IV presents the results on the effect of Amytal on 0, uptake. As shown in Expt. A, at zero day, Amytal at a concentration of I mM inhibited endogenous oxidation 49 %. Despite the fact that glucose stimulated 0, uptake of uncultured cells, the inhibition by amytal in the presence of glucose was much greater, increasing as it did to yo ‘$6, and the value of 0, uptake in the presence of gluco se 2nd Amytal was even lower than that in the presence of Amytal alone. Pzlmitate at a concentration of 1 m gave a greater stimulation to 0, aptake by uncultured cells. However, in contrast with the effect of glucose, pahmitate stimulation does not change the effect of Amytal, which inhibits 0, uptake to the same extent as in the endogenous oxidation. In five experiments the addition

of glucose

to an Amytal-inhibited

primary

culture increased the inhibition of 0, uptake despite the fact that addition of glucose to non-inhibited cells increases 0, uptake. In these same experiments addition of palmitate, to Amytal-inhibited cells, increased 0, uptake. One interpretation of these results is that glucose shifts met2bolism through a flavin that is more sensitive to amytal inhibition. If this is true, then the effect of palmitate in stimulating 0, uptake but not changing the Amytal effect would indicate that the endogenous metabolism is going through the same flavin as is involved in the oxidation of palmitate. This would indicate that fat is the endogenoas substrate and supports the conclusion reached from R. &_ measurements. In 8 days the picture changes. It can be seen that in Expts. I3 and C, Table IV, the 0, uptake decreases, glucose now inhibits 0, uptake and the ability of palmitate to stimulate 0, uptake is Biochivn.

Biophys.

Acta,

86 (x964)

74-80

METABOLIC

79

SHIFTS IN CULTURED CELLS

lost. The inhibition by Amytal is greater and only a slight increase in inhibition is observed following the addition of glucose. At 15 or 18 days in culture, the absolute value of endogenous inhibition by amytal is greater and essentially the same as in the presence of glucose or palmitate. The absolute value of endogenous inhibition by Amytal in older cells is close to, that given by glucose plus Amytal at zero day. One may reason that, since the metabolism has shifted toward carbohydrate in the older cells, the inhibition by Amytal of the more sensitive flavin achieves a maximum effect and is no longer dependent upon further addition of glucose. Thus, addition of glucose would no longer cause any further increase in inhibition by Amytal. The loss of ability of palmitate to stimulate 0, uptake also supports the hypothesis that cells in culture have shifted from fat to carbohydrate metabolism. When palmitate loses its ability to stimulate 0, uptake, it also loses the ability to shift metabolism to a system less sensitive to Amytal. It is interesting to note that the loss in the ability of palmitate to stimulate 0, uptake occurs at about the same time as the Crabtree and Pasteur effects appear. The observation that pyruvate oxidation decreases to about the same extent as that of the endogenous oxidation after 14 days in culture may indicate that the overall capacity of the cell to respire has decreased with time in culture. Such changes might be caused by either a loss of mitochondria or by an alteration in mitochondrial function. Possible changes in mitochondria during culture are under investigation. TABLE EFFECTOF Concentration

E@.

A

Days

of additions:

in cultwe

0

18

B

0

8

‘5

C

0

8

I5

AMYTALON

IV 0,

UPTAKE

glucose, 0.02 M; palmitate,

0.001 M; amytal, 0.001 M. 0, uptake

Additions

Inhibition Withwt

None Glucose Palmitate None Glucose Palmitate None Glucose Palmitate None Glucose Palmitate None Glucose Palmitate None Glucose Palmitate None Glucose Palmitate None Glucose Palmitate

Amvtal

8.6

17.0

49

7.6 15.6 1.6 I.1 I.5

70 45

49 68 57 68

9.0 II.2

II.7 8.1 15.0 5.6 3.3 5.5 3.5 3.0 3.1

22.0

12.3

24.9 28.4 8.2 6.0 8.2 23.0 25.3 35.0 17.6 12.6 17.6 12.0

24.6 30.5 13.5 IO.9 12.8 9.0 6.5 9.0

Biochim.

(%)

With Amytal

IO.1

16.5 4.2 2.8 3.7 2.2 1.65 2.3

Biophys.

Acta,

80

81 82

I$ 71 67 72 44 59 46 69 74 71 75 75 75

86 (1964)

74-8~

ACXNOWLEDGEMEXTS

We would like to acknowledge

the expert techcical

assistance of Mrs. 3. FARLEY.

This work was supper-ted in part by research grant Wo. A-2135 from the Kational Institutes of Health, U.S. Public Health Service, and -under corrtract Ko. AT-CM-~GEN-IZ

between

the U.S. Atomic

Energy

Commission

and the University

of Cali-

fornia, Los Angeles. REFERENCES 1 I. RARARY AND B. FARLEY, Exptl. Cell Res., 29 (1963) 451. H. KUR~MITS~ AND I.HARARY, Biochim. Biophys. .4cta, 86 (1964)65 3 H. EAGLE, Sciew.ce, 122 (195;)501. 4P. I.MARCUS, S. J. CIECIUR~AND T.T. PucK,~. I?‘x$tl.l~ed., 103 (1956)273. 5 A. L. LEHNINGER,]. Baol.Chem., 165 (1946) 131. 6 G. G. OLSOX,Clip.Chem, 8 (1962) I. 7 E. W. CRUICKSHANK,Physiol. Rev., 16 (1936) .597. * iV. B. VISSCHER,PIT. Sot. Exptl. Med., 38 (1938) 323. 9 R. J, BING, A. SIEGEL, I. CNGAR AND M. GILBERT.Am.J. Med., IG (Igj+) joz$. 10 J. C. SCOTT,1,. J.FINKELSTEINAND J. J. SPITZER,~?~.]. Physaol., 203 (1962) 482. ‘1 Ciba Fougzd. Sy~p., Regulation Cell Metab., Little, Bro\vn, Boston,1959,p. 205. I2J. VAN EYS, in D. M. BONXER, Co&ml Mechmisms in Cellular Processes, Ronald Press,New York, 1961, p. 141. 2

Biochim?.

Biophys.

&‘icta, 86 (1964)

74-80