Deuterium oxide stimulates glucose metabolism and inhibits lipolysis in rat epididymal adipose tissue

BIOCHEMICAL

Vol. 64, No. 1, 1975

DEUTERIUM AND INHIBITS

OXIDE

AND BIOPHYSICAL

STIMULATES

LIPOLYSIS

RESEARCH COMMUNICATIONS

GLUCOSE

METABOLISM

IN RAT EPIDIDYMAL

Richard

ADIPOSE

TISSUE

Schimmel

Department of Physiology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15261 Received

March

21,197s

Summary: Incubation of segments of rat epididymal adipose tissue in media prepared with deuterium oxide results in increased glucose oxidation, increased lipogenesis, accelerated sugar transport and decreased lipolysis in response to epinephrine or theophylline. In view of the well documented action of heavy water to “stabilize” cytoplasmic microtubules, the foregoing observations are in support of a link between cytoplasmic microtubules and metabolic process in adipose tissue.

In recent various

years

tissues

A function the outward stimulated

the physiological

has been the subject

for microtubules movement lipogenesis

microtubules.

(5) are inhibited to gain a study

tissue

function,

on adipose

tissue

was initiated. to “stabilize”

dependent

upon

microtubules

research

tissue

of free fatty acids

In an effort

it has been found

of much

in adipose

in adipose

cell functions

role of cytoplasmic

and speculation

has been postulated

from

adipose

tissue

by colchicine,

additional

insight

because

known

to disrupt

into the role of microtubules

for this

cytoplasmic

microtubules

these cellular

organelles

347

(l- 3) .

(4) and insulin

a drug

of the effects of deuterium

D20 was selected

in

oxide study

because

and interfere (6,7)

(D20)

with

Vol. 64, No. 1,1975

Materials

and

Normal from

BIOCHEMICAL

Methods

male rats

Zivic-Miller

by cervical Segments

(160-200g)

Lab,

Inc.

dislocation weighing

of the Sprague-Dawley

The animals

of Krebs-Ringer

between

indicated

among

of 02:

V) , insulin

C02,

and glucose

in the text.

(pH, 95/S

were

14C-glucose

7.4) (V/V)

51.2 mci/mMole)

indicated

of 0,20Pci/ml.

and used at various constant

for D20

to prepare

concentrations (0.2 x 10

-14

D20 containing

and again a standard

examined

with

pH meter

by the method

transport

mechanism

with

transport

Results

and Discussion I shows

(12,13)

the dissociation

it is not possible

in many tissues,

simultaneously

reason,

once with

the effects

pD = pH

was measured

using

(8) . Incorporation as described

(10) and free fatty acids

on the effects which

where

from Sigma

and CO2 was determined

of D-xylose, glucose

45 mci/mMole)

For this

conditions:

(Sigma,

at concentrations

a pD and pOD equal

enzymatically

(11) . In studies

albumin

Because

solution.

regions

1.0 ml

was present

that pD = pH + 0.4

into lipid

accumulation

of sugar

Table

water

was measured

of Dole

the intracellular

having

and the relation

(9) , glycerol

in some media

The pD of D20 solutions

from glucose

serum

position

distal

for 60 minutes

D20 was obtained

two different

pOD = pOH.

of radioactivity by Bray

under

. Bovine

) is less than that of H20,

to the pH and pOH of a comparable of D20 were

and incubated

up to 99% (V/V)

solutions

excised.

containing

in the 1 (ICN,

or in the 6 (Schwarz-Mann, at a concentration

rapidly

vessels

purchased

sacrificed

cut from the thin

present

labelled

were

overnight,

fat bodies

incubation

buffer

strain

fasted

30 and 70 mg were

bicarbonate

an atmosphere

Fraction

were

and the two epididymal

of the fat pads and distributed

under

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of D20 on glucose

appears

to share

transport,

a common

was used as an indication

.

the effects of D20 on lipolysis.

348

In the absence

of albumin

BIOCHEMICAL

Vol. 64, No. 1,1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Effects

of Deuterium in Epididymal

Oxide Adipose

on Lipolysis Tissue

Measurement Additions Incubation

to Buffer

Glycerol

Release Da0

HzO 1.

2.

3.

None epinephrine, epinephrine, theophylline,

1.3 3.1 4.6 5.8

O.O5pg/ml 0. lOpg/ml O.lOmg/ml

Glucose epinephrine epinephrine,

, O.O5pg/ml O.lO~g/ml

Glucose & albumin epinephrine , 0.05 epinephrine, 0.10

FFA Release

+ 0.3 50.4 2 0.5 +_ 0.7

1.1 to.2 1.3 + 0.3* 1.8 + 0.3* 3.150.4”

1.0 t 0.3 3.3 + 0.3 4.5 + 0.5

1.2 LO.3 2.6 + 0.2 3.7 + 0.4

1.8 + 0.3 4.0 + 0.5 7.2 c 0.8

1.6 +_0.3* 1.8 +0.3* 3.2 +0.4*

Tissue

DzO

H-20

3.8 + 0.5 5.8TO.7 7.1 -+ 1.1

FFA GO

Ha0

0.2 + 0.4* 1.2 + 0.5* 0.8 + 0.4*

Segments of rat epididymal adipose tissue were incubated in Krebs-Ringer bicarbonate buffer prepared with Hz0 or with SO% (V/V) D20 (pH : pD = 7.41 for 60 minutes. Glucose was presented where indicated at a concentration of 1 mg/ml: bovine serum albumin at 40 mg/ml. The units of glycerol and free fatty acid release are pmoles/g tissue wet wt-hr: tissue free fatty acid content Each value represents the mean + SE of 12 tissues. is expressed as pmoles/g tissue wet wt.

* buffer:

Significantly p < .05.

and glucose,

different

from

corresponding

a 50% solution

of epinephrine

values

obtained

of D20 markedly

or theophylline.

inhibited

When glucose

although

acids

suggesting

free fatty acids

remained

was accelerated

effect of D20 was also evident all subsequent

depressed

data were

adjusted

to 7.4 (POD,

in media

with

in the presence in medium

obtained

7.3) ; however,

pOD adjusted

virtually

to 6.6 (pD,

349

8.1) .

effects

of free fatty

that esterification

of D20.

identical

of

The antilipolytic

albumin.

incubated

in Ha

the antilipolytic

accumulation

containing

from tissues

incubated

the lipolytic

was present,

effect of D20 was much less pronounced in the tissues

from tissues

These,

in media results

with were

and pD obtained

-

BIOCHEMICAL

Vol. 64, No. 1, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE Effect of Specifically

of Deuterium Oxide on Metabolism Labelled T1”Cl - glucose by Adipose

Incorporation

GO o/o

Insulin

II

of 14C into

Fatty

co2

(CPM/mg-hr)

acids

GlycerideGlycerol

mp/ml

0 33 66 99 0 33 66 99

10.8 2 1.5 10.5 + 2.1 19.6 + 1.2* 30.1 + 2.5* 18.8 + 2.1t 23.6 + 2.7t 34.6 t 4.1*? 33.3 + 2.1*

1.50 1.50 3.50 3.50

0 33 66 99 0 33 66 99

6.02 1.9 6.9 + 0.8 9.8 + l.O* 11.8 t 0.8* 8.6+0.9? 9.22 0.5t 11.3 + 1.1* 12.1 + 1.3*

I. 50 3.50 3.50 3.50

[Glucose-l 1.12 0.1 1.12 0.1 3.0 & 0.5* 7.6 5 1.3* 4.2 2 l.lt 5.9 + 1.1t 9.0 + 1.2* 9.3 + 1.1*

‘“Cl

[Glucose-6

‘“Cl

3.3 4.6 11.2 25.2 22.1 30.3 29.1 31.9

5 0.1 + 1.1 + 1.3* + 1.9* 2 1.4t +7.3t +2.1*? t 2.4*

Total

10.6 + 1.2 18.0 + 1.9* 28.5+3.1* 35.6 + 3.8* 21.4 23.1.t 25.3 + 3.8t 33.3 + 3.8* 41.9 + 5.1*

22.5 29.6+ 51.1+ 73.3 44.4 54.8 76.9 81.5

t + 2 t +

21.7 26.3 29.2 38.1 34.3 35.5 35.6 45.3

31.0+ 37.8 50.2 75.1& 65.02 75.0t 76.0 89.3

4.0 4.2 4.1* 8.8* 6.7t 8.1? + 8.2t +lO.O*

+ 2.3 + 2.8 + 2.8 +3.1* + 4.4t 24.2 + 3.9 + 5.2

Segments of rat epididymal adipose tissue were incubated nate buffer containing various amounts of DsO for 60 minutes. at a concentration of 1 mg/ml: %-glucose at a concentration value represents the mean -+ SE of 12 tissues.

*

Significantly

t

Significantly different present: p< .05.

different

The metabolism are incubated II)

. This

from

for the greater

in Hz0 value

appears

to be increased

prepared glucose

esterification

obtained

corresponding

of glucose

in media accelerated

from value

with

Tissue

metabolism

of free fatty acids

350

i 2

- buffer with

when

amounts

caused

2.8 2.8* 7.2* 9.1* 5.2t 5.lt 6.8*t 10.2*

in Krebs-Ringer Glucose was of 0.20 pci/ml.

obtained

increasing

+

tissue

of D20

by D20 probably

in tissues

incubated

no insulin

segments (Table accounts in D20

bicarbo present Each

BIOCHEMICAL

Vol. 64, No. 1, 1975

(Table

I) . Increased

to co2,

metabolism

enhanced

Conversion than glucose-6

When insulin

l*C to l*COz

pathway

i mplying

a greater

a pattern

in the presence

insulin

to a greater

extent

of glucose

through

flux

of glucose

metabolism

In addition,

failed

oxidation

of glyceride-glycerol.

in media

as the concentration

of 99% D20,

in enhanced

incubated

by D20 was evident.

decreased

formation

was increased

in tissues

was present,

to that produced

was reflected

and increased

l*C to l*C02,

the pentose-shunt

to insulin

of glucose

lipogenesis

of glucose-l

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

prepared

from D20.

qualitatively

the tissue

similar

responses

of D20 in the medium

increased;

to accelerate

metabolism

glucose

significantly. Insulin

increases

the transport

glucose

of the sugar

metabolism

into adipocytes

that D20 may have a similar in the xylose xylose

in increased

xylose

was measured, The salient of rat epididymal effects upon

sugar

of D20.

observation

reported

herein

adipose

tissue

by exposure glucose

prepared which

to insulin.

with

of these data requires

consideration cellular

incubated

in D20.

of segments

heavy

water

exerts

effects of

in response

351

were

metabolism

These

lipolysis

the best-known

also resulted

similar

increased

To date,

space

are remarkably

oxidation,

and decreased

tissues

of

of tissues

glucose

is that incubation

in buffers

of the tissue

to insulin

as was seen when

was lost when

entry

the sucrose

of tissues

however,

increase

incubation

in either

response

increased

transport

Exposure

III show

an accelerated

In contrast,

change

by accelerating

a dose-dependent

the insulin

caused

Interpretation action

transport;

the metabolism

changes

D20 include

compartment.

content.

in part,

(14) . The data in Table

suggesting

in a measurable

water

tissue,

D20 produced

tissues,

into the intracellular

or the total tissue

those

action.

space of adipose

in D20 did not result

in adipose

metabolic

to

lipogenesis

, accelerated

to lipolytic

agents.

action

of the mechanism of heavy

water

of is

BIOCHEMICAL

Vol. 64, No. 1,1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

of [‘“Cl

J&O (o/o) 0 33 66 100 0 33 66 100

- Xylose

Effects of Deuterium and [‘“Cl - Sucrose

Insulin (w/ml)

Total water (~l/lOOmg)

252 24 + 262 23 + 28 + 27 + 26 + 26+

0.50 0.50 0.50 0.50

III

Oxide on the Distribution in Water of Epididymal

Xylose

1 2 1 2 2 2 3 2

space ( % of Total

66.624.2 71.12 82.9 + 84.3 + 86.lL 84,5 + 89.4 f. 90.7+8.2

Adipose

Tissue

Sucrose

Space

water)

58.226.2 59.12 5.1 61.2 ~fi 5.8 60.425.1 60.1 24.9 63.2 + 6.1 61.4 + 6.2 59.5 +5.5

6.1** 6.6* 6.8* 5.2t 4. It 5.8

Segments of rat epididymal adipose tissue were incubated in KrebsRinger bicarbonate buffer containing various amounts of DzO for 60 minutes. 14C-Xylose or 14C-Sucrose were present at concentrations of 1.0 mg/m‘ Each value represents the mean + SE of 16 tissues. and 0.20 pci/ml.

*

Significantly

different

from value

t Significantly different from insulin present, p’. 05, **Mean

to foster

difference,

corresponding

4.5 + 1.9:

the polymerization

It follows

that the metabolic

alteration

in the microtubular

obtained

value

- buffer:

obtained

p < .05.

with

no

PC .05.

and increase

the strength

effects of D20 could system

in Hz0

in adipose

352

of microtubules

result

from a functional

tissue

cells

(6,7) .

and that microtubule

BIOCHEMICAL

Vol. 64, No. 1, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

assembly may, in some as yet undefined manner be an essential component in the regulation of metabolic activity in adipose tissue cells. This explanation is in accord with the view advanced independently from this laboratory

(4)

and by Soifer et al (5) linking metabolic processes in adipose tissue cells with microtubules.

It should be noted that Soifer et al (5) presented evidence

that insulin stimulates the formation of microtubules in adipocytes , an action similar to that attributed to D20. However, D20 exerts other general actions on tissues incubated in vitro (15-18) and in view of the apparent diversity

of action of D20 it may be premature

to ascribe its actions on adipose tissues to assembly of microtubules,

Acknowledgements: The excellent technical assistance rendered by Mr. Dennis McKeag is greatly acknowledged. This research was supported by a grant from the National Institutes of Health, AA4 15466.

References

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Vol. 64, No. 1,1975

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BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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