Effects of biguanides on fatty acid and glucose oxidation in muscle

Pharmacological Research Communications, VoL 6, No. 3, 1974 EFFECTS

OF B I G U A N I D E S

ON F A T T Y

OXIDATION

GoU. Corsini~

ACID

253

AND G L U C O S E

IN ~ S C L E .

Fo Sirigu~

P. Tagliamonte

and S. Muntoni

Department of Pharmacology and Chemotherapy, University of Cagliari, 09100 Cagliari~ Italy and 2 nd Division of Medicine and Center for Metabolic Diseases and Arteriosclerosis~ Municipal Hospital of Cagliari, 09100 Cagliari~ Italy 2eceived 18 September 1973 SUMMARY

Non-toxic

formin depress the

concentrations

Moreover,

the inhibition

tion by unlabelled palmitic biguanides,

or met-

14C02 production from pal~itic-U-14C by rat diaphragm homo-

acid~ but not from glucose-U-14C~ genateo

of phenformJn

of glucose-U-14C

oxida-

acid is partly removed by

These data demonstrate

that biguanides

pro-

duce their effects on glucose m e t a b o l i s m through depression of fatty acid oxidation.

There is a considerable, that the effects of biguanides secondary to inhibition 1968~ Muntoni

et alo~

although indirect

on glucose m e t a b o l i s m

of fatty acid oxidation

1969,

evidence

1970; Muntoni

are

(Mnntoni,

and Sirigu,

The aim of the present investigations

1971)o

was to achieve

direct evidence of such a m e c h a n i s m of actiono

MATERIALS AND I~THOD,S

Male albino rats of the Wistar

strain (200 g) were housed at a temperature access to food and water

ad libitum.

Animals

of 22 ° C, with were killed by

Pharmacoiogica/ Research Communications,

254

Vol. 6, ~1o. 3, 7974

decapitation and the diaphragms rapidly removed and homogenized in 5 ml of Krebs Ringer calcium-free phosphate buffer (pH 7,4) in a Potter homogenizer, of homogenate~

Protein concentration

assayed by biuret method~ varied between 8

and 12 mg/lO0 ml, The homogenate (I ml) was incubated in Warburg-like flasks adapted to collect free C02: the central tube contained a strip of adsorbant paper impregnated with hyamine hydroxide (I N)o Palmitic-14C

acid (OOO5 ~C) unifor-

mly labelled (NEN) and dissolved in dimethyl-formamide (lO ~i) was added to the incubation mixture,

Glucose-14C

(0,04 ~C) uniformly labelled (NEN) was in aqueous solution (50 ~I), Phenethyl-biguanide

hydrochloride

(Aldrich Chem,

Co,)~ or dimethyl-biguanide, hydrochloride (Aron Lab,~ Suresnes~ France) in aqueous solution were added where indicated, The incuba~ion~ performed at 35 ° C in agitation bath for 30 minu~es~ was stoppe d by addition of I ml sulphuric acid (4 N)~ and the samples were allowed to equilibrate the CO 2 for 2 hours at room temperature,

After this peri~ d the paper

was tested for radioactivity in a Packard Scintillation Counter using an appropriate scintillation liquid,

RESULTS

The amount of 14CO2. produced from oxidation" of

palmitic-U-14C

acid by rat diaphragm homogenate in glucose-

-free medium was l o w e r e d b y tions:

various phenformin concentra-

Io-bM caused 15 pe r cent inhibition~ while 10 -4 and

IO-3M inhibited respectively by 32 and 74 per cent (Table I), The changes caused ,by phenformin on glucose-U-14C oxidation in t h e absence of exogenous palmitic acid are shown in Table 2:

Io-bM ,phenformin did not cause any changej

Pharmacological Research Communications, VoL 6, No.. 3, 1974 TABLE

255

1,

Effect of phenformin on palmitic-U-14C acid oxidation by rat diaphragm homogenate (CPM 14C02 per IO mg protein) ,,

Shbstr ate

,,,,

,,

.,

,

J

•

.,,

,

,

CPM 14C02 _+ S.E, Per cent changes after 30 rain.

Phenformin

Palmitic-U- 14C acid IO-4M

1,47'4 + 59

id,

Io-SM

1,253

+ 86

-

15

ido

IO-4M

1,007

-+ 45

-

32

*

id,

IO-3M

+ ,5

-

74

**

,,,

,

,,,

Significance levels : * p <0o05

** p < O , O 1

TABLE 2,

Effect of phenformin on glucose-U-14C oxidation by rat diaphragm homogenate (CPM 14C02 per lO,mg protein) ,,.

.

.

,,,.

_

_

Substrate

Phenformin

!ucose-U- 14C

CPM 14C02 ~ SoB, P e r cent c h a n g e s after 30 min,

2,475 + 114

I0-3M id,

IO-SM

2,433

+

I06

id,

IO-4M

2,284

+

170

ido

IO-3M

Significance levels: w~ p
545 +.. 37

-

8

- 78 **

Pharmacological Research Communications, VoL 6, No. 3, 1974

256

TABLE 3 • Effect of phenfornin on palmitic-U-14C by rat diaphragm homogenate

acid oxidation

(CPM 14C02 per 10 mg protein)

in the presence of glucose ,.

,, , ,

,

CPM 14CO 2 _+ S,E, after 30 ~in.

Phenformin

Substr ares

q

,

,

Per cent changes

,

Palmitic-U- 14C acid IO-4M plus Glucose IO-3M id.

1,421 _+ 84

IO-4M

Significance levels :

1,O20 + 47

- 28 *

* p <0,05

TABLE 4 . Effect of phenformin on glucose-U-14C by rat diaphragm homogenate

(CPM 14CO 2 per iO mg protein)

in the presence of palmitic m

Substr ares

m

I I

I

ml

CPM

Phenformin

II

I

I

14C02 + S.E.

acid II

m I

II

II

I

ml

Per cent changes

1 , 1 4 9 + 53

lO-4M

1 ~ 6 8 5 + 42 I

Significance

II

after 30 min~

Glucose-U-14C IO-3M plus Palmitic acid IO-4M id,

I

oxidation

levels :

I

i

II

-X-. p < O o O 1

I

+ 46.7

**

Pharmacological Research Communications,

VoL 6, No. 3, 1974

257

IO-4M inhibited

by 8 per cent s 'while IO-3M inhibited

78 per cent the

14C02 productiono

The following

experiments

aimed to check whether

same changes were caused by phenformin from palmitic-U-14C of the alternative Glucose

unlabelled

rateo

~ in the presence

substrate,

glucose.

32 per cent)

medium caused a marked decrease

Table 2 in comparison

with

under this experimental

I)

was about the

in the two caseso

palmitic

acid added to the

(54 per cent)

rate (see control

Table

of inhibition

on 14C02 production

On the contrary s unlabelled

values,

in glucose-

2,475 CPM, in

1,149 CPM in Table 4), Besides~

condition

IO-4M phenformin

by 46°7 per cent the glucose-U-14C as CO 2 produced

of 14C02 produced

(1,474 CPM,

Also the magnitude

same (28 per cent versus

oxidation

palmitic

acid was about the same in the presence

caused by IO-4M phenformin

Similar

_14,.

Table 3) as in the absence

of unlabelled

-U-14C

U

In fact s the amount

from palmitic-U-14C (1,421 CPM~

_

acid or glucose

the

on 14C02 production

added to ~he medium did not influence

acid oxidation

by

oxidation

rate,

increased expressed

(Table 4),

effects~

not reported

duced by 10-4M metformin

in the Tables,

on palmitic

were pro-

acid and glucose

oxi-

dation.

DISCUSSION

Our present

the CO 2 production genateo

from palmitic

The magnitude

concentration discussed

data show that phenformin

acid in rat diaphragm

homo-

of such an effect is related to the

of the drug.

elsewhere

lowers

However,

(Muntoni,

a good deal of arguments

1973) suggest that 10 -3 M

Pharmacological R.~search Communications,

258

phenformin is a toxic concentration,

Therefore~

VoL 6, No. 3, 1974

inhibition

of oxidative p h o s p h o r i l a t i o n produced by phenformin (Pressman,

1963;

Sch~fer,

1969)

is, in our opinion,

a toxic effect, which

can account for the dramatic fall in CO 2 p r o d u c t i o n from both palmitic acid and glucose, On the other hand 3 phenformin concentrations IO-SM can be regarded as non-toxic

(Muntoni, 1973).

10 -4 to

(except for in guinea pig)

Therefore their effects are likely to be

of "pharmacological"

nature,

w h e n the latter concentrations

are used$

from palmitic acids.but not from glucoses

CO 2 production

is depressed,

This

fact rules out any impairment of oxidative p h o s p h o r i l a t i o n and rather suggests a specific i n h i b i t i o n of palmitic

acid

oxidation, Moreover,

enhanced CO 2 p r o d u c t i o n from glucose does not

appear as a direct effect of phenformin,

In fact, when glu-

cose is the only available substrate CO 2 production remains nearly u n a f f e c t e d by the drug, O n

the c o n t r a r y 3 when gluco-

se-U-14C is u t i l i z e d in. the presence of u n l a b e l l e d palmitic acid and hence its oxidation is depressed~ ces 14C02 production,

In other words~

p h e n f o r m i n enhan-

p h e n f o r m i n partly res-

tores glucose oxidation depressed by palmitic

acid: therefo-

re, increase in CO 2 p r o d u c t i o n from glucose is a consequence of depressed palmitic

acid oxidation,

W h e n m e t f o r m i n is used i n s t e a d of phenformin~

the same

effects are observed, In our opinionj these data represent the key point of the biguanide m e c h a n i s m of action,

On the one hand, they

agree well with those of Randle et al, (1965), who found a 70 per cent i n h i b i t i o n of pyruvate oxidation by palmitate

Pharmacological Research Communications, in rat diaphragm. that biguanides

VoL 6, No. 3, 1974

On the other hand,

partly remove

259

our data demonstrate

such an inhibition.

This ef-

fect recalls the one produced by 2-bromostearate,

an inhi-

bitor of fatty acid oxidations the insulin effectiveness tion of glucose-U-14C rats (Randle,

which was found to restore

on uptake,

mcbabolism

and oxida-

in m y o c a r d i u m from alloxan-diabetic

1969).

The concept still debated$

of the glucose

fatty acid cycle s although

was b a s e d on sound experimental

evidence

de-

monstrating that increased rates of fatty acid oxidation impair uptake, the presence

glycolysis

and oxidation

of insulin in muscle,

genesis in liver In obesity

(Randle et al.,

of glucose

and stimulate

even in

gluconeo-

1965).

and maturity onset diabetes increased

availa-

bility and oxidation rates of fatty acids are thought to contribute to impaired glucose tolerance, with secondary hyperinsulinemia, (Randle et alo,

196i~ R u d e r m a n

hand s in the same metabolic

and enhanced gluconeo genesis

et al.,

of glucose

(Muntoni

et al.,

1969). On the other

diseases biguanides

to increase glucose uptake by muscle to improve glucose tolerance

insulin-resistance

are known

(Butterfield,

(Butterfield,

1968)

and K ~ralue

1968), to enhance glucose

tion to CO 2 (Searle and Cavalieri~

1968),

increased levels of plasma insulin

(Grodsky et al.,

Moreover s low phenformin

concentrations

bit hepatic g l u c o n e o g e n esis rat, in vivo Clearly~

(Connon,

1968),

and to lower the

were

in diabetic,

oxida-

1963).

shown to inhi-

but not in normal

1971)o

all these metabolic

effects

of biguanides

are

contrary to those produced by high rates of long chain fatty

Pharmacological Research Communications, Vol. 6, No. 3, 1974

260

acid oxidation in muscle and liver.

Therefore, depression

of fatty acid oxidation rates can simultaneously account for all the above mentioned biguanide effects, This 'possibility was firstly suggested and the definition of biguanides as " d r u ~ s

(Munt0ni, 1968)~

acting on the Randle cycle" was proPOsed later it received indirect experimental

evidence (Muntoni et al.,

19693 1970).

Our present 'data

provide the ultimate direct demonstration of such a mechanism of action. How biguanides specifically inhibit palmitic acid oxidation is now under investigation. The starting-point for this was the possibility~

(Muntoni, 1973),

suggested and discussed elsewhere

that biguanides affect the carnitine-depen-

dent steps of fatty acid metabolism and~ in" particular, the long chain fatty acid transport across mitochondrial membrane. Preliminary results obtained with the NMR technique showed that phenformin, metformin and buformin interact with carnitine~ in vitro~ producing complexes with it (Crisponi et al.,

1973). These investigations are still in progress4

In conclusion,

"non-toxic" concentrations of biguanides

selectively inhibit palmitic acid oxidation.

This effect re-

presents the essence of biguanide mechanism of action, "inasmuch as it accounts for the other effects on glucose metabolism and insulin effectiveness on obesity and maturity onset diabetes mellitus.

REFERENCES

Butterfield, W.J.H. Corm,n, J.J. (1971) Southampton

(1968)

Ann. N. Y. Acad. Sci. 148, 724 7th Ann. Meeting Europ. Ass. Diabetes,

Pharmacological Research Communications, Vol. 6, No. 3, 1974

Crisponi~ Go~ Lai, A., Rossetti, Z.L., Muntoni, So~ Corsini~ GoUo, Tagliamonte~ P. and Sirigu+ F. (1973) submitted for publication Grodsky, GoM.~ Karam~ J.H., Pavlatos, FoCo and Forsham, Po (1963) Metabolism 1_~2, 278 Muntoni~ S. (1968) PrOCo Inferno Syrup. Antidiabetic Biguanides, Rimini M~ntoni~ So (1973) Advano Lipid Res., in press Muntoni~ So, and Sirigu, F. (1971) Rasso Med. Sarda ~ , 241 M~ntoni, So ~ Sirig~, F. ~ F!oris ~ Mo and Boero, A. (1968) Min. Medo Giuliana 8, 256 Muntoni, So, Duce, Mo and Corsini, G.U. (1969) XV Congro Soc. Ito Farmacol., Milano Muntoni, So, Duce, Mo and Corsini, GoU0 (1970) Life Scio 9 (Po IX), 241 Pressman, B°Co (1963) Jo Biolo Chem° 23_~8, 401 Randle, PoJ. (1969) Nature (Lond.) 2Zl, 777 Randle~ P°J.~ Garland, P.B., Newsholme, EoA. and Hales~ C.N. (1965) Anno No Y° Acado Sci. 131, 324 Ruderman, N©B., Toews, Co,. and IShafrir, E° (1969) Arch. Inferno Med. 123, 299 Sch~fer, Go (1969) Biochimo Biophys. Acta ~ , 334 Searle, G.Lo and Cavalieri, R.R. (1968) Ann. No Y. Acad° Scio 148~ 734

261