Metabolic effects of buformin on the isolated perfused rat hindlimb during electrical stimulation

Pharmacological Research Communications,. Vol. 9, No. 8, 1977

711

METABOLIC EFFECTS OF BUFORMIN ON THE ISOLATED PERFUSED RAT HINDLIMB DURING ELECTRICAL STIMULATION. P. Strohfeldt,

U. Strubel-Obermaier

Department of Internal Medicine, Homburg/Saar,

University of Saarland Germany

Received5 March 1977 Oral pretreatment of normal rats with a blood sugar lowering dose of buformin

(15o mg/kg Per day)

increases lactate

production of contracting skeletal muscle tissue without any additional stimulating effect on the enhanced glucose uptake. These date together with those presented elsewhere and Strubel-Obermaier,1976)

(Strohfeldt

demonstrate that biguanides act un-

specifically on skeletal muscle tissue including inhibition of oxidative phosphorylation.

A useful drug action on skeletal mus-

cle tissue is in doubt. Introduction

The mode of action of the biguanides

being discussed.

is still

In particular this is the case for the peri-

pheral action of the drug. We found that butyl-biguanide was unable to increase the glucose uptake of the resting skeletal muscle using the isolated perfused rat hindlimb.

Furthermore

we could not observe an insulin potentiating effect of buformin on glucose uptake of the skeletal muscle

(Strohfeldt et al.,

1975). Our results are in contrast with findings obtained in man or with in vlvo models ai.,1961;

(Butterfield,1968;

Losert et a1.,1972)

Creutzfeldt et

or with the incubated diaphragm

(Bolinger et a1.,196o), but agree in part with the results of investigations based on the perfused rat heart as an experimental model

(Williamson et ai.,1963). In order to make the used

experimental system more sensitive to metabolic changes caused

Pharmacological Research Communications, VoL 9, No. 8, 1977

712

by the drug we investigated ing dose of buformin glycerol

release

electrical

the effect of a blood

glucose uptake, lactate p r o d u c t i o n and

on

by the isolated

stimulation.

on FFA c o n c e n t r a t i o n

perfused, rat hindlimb

In addition

the influence

in the perfusate

~at_eria_isand Methods

was given addition animals

via a stomach

the p e r f u s a t e received

cal p r e p a r a t i o n bed elsewhere

contained

and perfusion

I u g / m l buformin.

technigue

(S£rohfeldt,1973). containing

freshly w a s h e d

erythrocytes

present

carbogen.

at the b e g i n n i n g

was about

nerves was obtained

toms at a rate of 4/s. contractions

sue and are e x p r e s e e d statistical

R@su!t~

tube

and

applied

for

throughout

of glucose,

lactate,

the same a s d e s c r i b e d

et al. ,1974).

by e r y t h r o c y t e s

as umol/gxh.

and

(Ruderman et a l . , 1 9 7 1 ; B e r g e r

and FFA in the m e d i u m were for g l y c o l y s i s

lactate

(Strohfeldt

were o b t a i n e d

for d e t e r m i n a t i o n

(Strohfeldt,1973; Strohfeldt

5 , 5 mM

was 7 V. With this m e t h o d

of the m u s c l e s

The m e t h o d s

were

of both the sciatic

Square wave pulses were

et ai.,1976).

corrected

substrates

ml)

in the arterial

electrodes

p e r i o d of 3o m i n u t e s

where

serum albumin,

experiment:

stimulation

The v o l t a g e

of surgi-

g haemoglobin/too

the pressure

the p e r f u s i o n

glycerol

(lo-ll

The following

by p l a t i n u m

Strubel-Obermaier,1976). constant

4 g% bovine

Ioo m m Hg. Electrical

The control

m e d i u m was com-

1,6 ± o,o5 m M endogenous

The flow rate w a s 2o ml/min,

In

were the same as descri-

of each p e r f u s i o n

glucose, o,15 ~,! pyruvate, + 0,8 - 0,03 mM FFA.

was

the last dose

The procedure

The p e r f u s i o n

posed of KHB-buffer,

and was gassed w i t h

for 4 days,

saline.

16o -

(15o mg/kg)

the start of £he experiment.

physiological

human

of buformin

rats w e i g h i n g

Buformin

tube daily

2,5 hours before

during

was measured.

Male S p r a g u e - D a w l e y

18o g and fed ad libitum were used. administered

sugar lower-

The results

in the absence

The W i l c o x o n

elseare

of tis-

test was ~sed for

analysis.

The results, that are summarized in the table show that

the resting perfused rat hindlimb utilized glucose and FFA. Electrical stimulation significantly enhanced the glucose uptake whereas the decrease of FFA level was not significantly diffe-

Pharmacological Research Communications, Vo/. 9, No. 8, 1977 rent f o l l o w i n g rable uptake resting

lactate

of lactic

glucose

Effect

uptake,

electrical

lactate

by oral p r e t r e a t m e n t details

Experimental conditions

stimulation

acid took place.

of bilateral

hindlimb.

perfused

glycerol

was

release

on

a n d FFA

and its influence for several days.

and methods.

Lactate production ~mol/gxh

of

stimulation

(15o mg/kg)

see under m a t e r i a l s

Glucose uptake

a significant

of the experiments

rat hindlimb

with buformin

of the

The c o n c e n t r a t i o n

sciatic nerve

formation,

A conside-

in the perfusions

at the beginning

in the isolated

For further

of the perfused

acid occurred

During

in the medium

I :

uptake

of lactic

hindlimb.

accumulation

Table

stimulation

713

Glycerol release

FFA uptake

( X + S~ )

Resting n = 5

-4,6-+ot9

-2,2+0,6

+0,29_+0,08

-o, 18_+0,05

Stimulated n = 5

x -9,1+I,o

x +5,8+0,9

+0,30-+0,08

-o, 59+0,1 5

x

Stimulated after buformin pretreatment n = 5 x : significantly (p < 0,05)

identical

different

in all the groups.

rat h i n d l i m b

was u n a f f e c t e d

Oral p r e t r e a t m e n t glucose

-9,1+1,9

The glycerol by e l e c t r i c a l

with buformin significantly

sed during p e r f u s i o n pretreatment,

+0,59_+o,05

from the c o r r e s p o n d i n g

release

+o ,89+0, 42

control

of the p e r f u s e d

stimulation.

had no effect on the e n h a n c e d

uptake of the e x e r c i s i n g

acid h o w e v e r was

release.

+13,6_+1,2

x

hindlimb.

stimulated.

of the stimulated

Formation

The FFA level

hindlimb

but there was no significant

of lactic increa-

after b u f o r m i n

change o~ the glycerol

714

Pharmacological Research Communications, Vol. 9, No. 8, 1977

Discussion

The rats were pretreated

dose of buformin that this

normal

(Strohfeldt et ai.,1972).

is already

experimental

a "toxic"

rats with buformin These

effect of buformin With

in normal

et al.,

creatine phosphate

skeletal muscle

tissue

(Strohfeldt

the metabolism

ced by electrical

contracting

per

consider

pretreatment

muscular

the intensity

tissue,

creatine

one has

rat hindlimb

of the workload

phosphate

it

induit

or glycogen

of the applied workload, (Strohfeldt

and

nor seems

at the end of the perfusion

drug itself acts on these parameters

on

pretreated

of the sciatic nerves

the ATP,

content of the muscle tissue

Obermaier,1976).

We therefore

isolated perfused

stimulation

parameters

were unaffec-

in both the biguanide

Using the

to monitor

in the resting

15o mg buformin/kg

the effect of buformin

to monitor

the concen-

for rats.

to apply identical workload

is not possible

an oral

30 and 175

Furthermore

and glycogen

et ai.,1975).

of perfused

the control group.

1975).

of rats with

as adequate

In order to investigate

therefore

after

and the oxygen consumption

ted by an oral pretreatment

as biochemical

of normal rats we ob-

effect of buformin

tration of ATP,

the used dosage

of

21o mg/kg per

for several days with dosages between

day for 4 days

the

oral pretreatment

rats only ~hen using

perfused hindlimb

mg/kg per day (Strohfeldt

reasonable

Following

even found an insulin ~otentiating

served no insulin potentiating pretreatment

lo%vering

up to 15o mg/kg per day can be regarded

authors

the isolated

(1972)

sugar

It may be objected

dose for the rat.

work of Losert et al.

as "non toxic".

day.

with a blood

period

because

the

and Strubel-

The only way of getting comparable

results

is to take careful notice of the experimental

condi-

tions. As was already demonstrated diabetic

with the hindlimbs

rats in the resting state

Strohfeldt,

Strubel-Obermaier

so non effect on the enhanced contracting m u s c u l a r tissue. duction

increased

ai.,1975).

and Kettl,1976),

Comparable

et al.,

and

1975;

buformin had al-

glucose uptake of the perfused On the other hand lactic acid pro-

significantly

contrast ~o the findings

(Strohfeldt

of normal

after buformin

in the resting

state

pretreatment (Strohfeldt et

results were obtained by Dietze

(1976)

in

Pharmacologica/ Research Communications, Vo/. 9, No. 8, 1977 with phenformin

treated maturity

forearm perfusion

technique,

715

onset diabetics

using the

~hen a workload was applied.

These date are in agreement with the view of Sch~fer He found that biguanides

unspecifically

tures and cause a positive mitochondrial

membranes

skeletal muscle

bind to membrane

shift of surface potentials.

this means an inhibition

uptake during ADP phosphorylati0n dence for this concept

(1976).

by biguanides.

is our observation

tissue of normal

greater breakdown of glycogen (Strohfeldt

A further

that in contracting

and creatine

phosphate without

cular contraction

after buformin

of glycerol

the muscle

tissue is derived

hydrolysZs

It is possible

after buformin inhibition

amount of the required

of endogenous

therefore

pretreatment

increases

studies on adipose

lipolysis

(Stone a. Brown,1968;

Only Lyngsoe et al.

(1972)

suggested

Tracer studies

utilization

(1974)

Whether

cannot be excluded.

Ditschuneit

are

increase

An inhibition of

in former perfusion

(Strohfeldt et ai.,1975) with normal rats

therapy.

et al.

Corsini

from homogenates

selectively

In vitro

in high

and

(1975)

et al.

and FFA

(1974) re-

of rat diaphragm muscle

inhibit palmitic

thereby partly restore glucose oxidation

experi-

(Losert et al.,

are also compatible with a decreased

ported results derived

bu-

et ai.,1968).

that biguanides

in man of Sch6nborn

during biguanide

that biguanides

tissue.

level of plasma FFA in star-

was already observed

in in vivo experiments

Stout et al.

study are due to an

after p h e n f o r m i n therapy.

ments with the resting hindlimb

1972).

This

while low concentrations

lipolysis because of an increased

described

energy of

(Carlson et

tissue have shown that biguanides

without effect

FFA utilization

a

that the changes observed

by the muscle

inhibit lipolysis

subjects

lipids

in the present

concentrations

ved normal

together with

from fatty acid oxidation.

of FFA utilization

formin directly

pretreatment

during mus-

from both an increased uptake of fatty acids

and a stimulated ai.,1971).

increased

release.

an increasing

fuel originates

with bu-

and Strubel-Obermaier,1976). of FFA in the perfusate

During exercise

evi-

rats there was a significantly

The concentration stimulation

For

of the proton

any change of the ATP content after oral pretreatment formin

struc--

acid oxidation

and

depressed by palmitic

Pharmacological Research Communications, Vol. 9, No. 8, 1977

716 acid.

Since there is evidence that the glucose fatty acid cycle

is also operating in skeletal muscle tissue Rennie et ai.,1976;

Maizels et ai.,1977),

(Cuendet et ai.,1975;

the glucose uptake of

the contracting muscle in our experiments must have been increased after buformin pretreatment, and his group is accepted.

if the hypothesis of Muntoni

It cannot be excluded that the rate

of increased glucose utilization was too small to be measured by the technics of this study. But in our opinion the presented data are compatible with an unspecific inhibitory effect of the drug on cellular respiration including decrease of both glucose and fatty acid oxidation. Biguanides have been shown to lower circulating lipid levels in some nondiabetic and diabetic humans

(Tzagournis et al.,

1968; Schwartz et ai.,1966;

Gustafson et ai.,1971;

1974; Kissebah et ai.,1974;

Lang et ai.,1973)

Stout et al.,

whereas the stu-

dies on the effect of biguanides on FFA levels were conflicting (Stout et ai.,1974).

Therefore the elevation of FFA concentra-

tion observed in the present study is probably without significance for the lipid lowering effect of the biguanides. In conclusion buformin increases the lactate production of contracting skeletal muscle tissue of normal rats without an additional stimulating effect on glucose uptake.

A specific drug

action on skeletal muscle tissue, which would be useful for the treatment of diabetes meilitus,-is put in doubt.

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