Br. J. Pharmacol. (1992), 105, 1009- 1013 '.

" Macmillan Press Ltd, 1992

Effect of metformin on glucose metabolism in the splanchnic

bed
'C.J. Bailey, Carol Wilcock & Caroline Day
Department of Pharmaceutical Sciences, Aston University, Birmingham B4 7ET
1 Use of the antihyperglycaemic agent, metformin, is often associated with a small rise in circulating
lactate. This study investigates the source of the lactate and examines the effect of metformin on glucose
metabolism by the intestine and liver of rats.
2 Changes in plasma glucose and lactate were measured in the inferior vena cava (IVC), hepatic portal
vein (HPV), hepatic vein (HV) and aorta (A) after intrajejunal administration of metformin (50 and
250mg kg-') without and with glucose (2 g kg-').
3 Metformin 250 mg kg-' reduced the hyperglycaemic response to a glucose challenge, associated with
a greater reduction of glucose concentrations in the HPV (average decrease of 33% at 60 and 120 min)
than at other sites.
4 Both doses of metformin increased lactate concentrations in the glucose-loaded state: the highest
concentration (2.5 fold increase) was recorded in the HPV 60 min after administration of 250 mg kg'
metformin, with a high lactate concentration persisting in the HV at 120 min. Metformin 250 mg kg'
also increased lactate concentrations in the basal state, with highest concentrations (2 fold increase) in
the HPV.
5 Two hours after intrajejunal administration of metformin, 50 mg kg-', rings of tissue from the small
intestine showed an average 22% decrease in glucose oxidation (['4C]-glucose conversion to '4CO2) and a
10% increase in lactate production. Since glucose metabolism in the gut is predominantly anaerobic,
metformin caused an overall 9.5% increase of intestinal glucose utilization.
6 Metformin, 10-6 and I0- mol 1', did not significantly alter glucose oxidation or lactate production
by isolated hepatocytes, but a very high concentration of metformin (102 mol 1') increased lactate
production by 60%.
7 The results support the view that metformin increased intestinal glucose utilization and lactate
production by the intestine. Under basal conditions there was net extraction of lactate by the liver but
not after an enteral glucose load.
Keywords: Metformin; glucose metabolism; lactate; splanchnic bed; liver; intestine

Introduction
Metformin (dimethylbiguanide) is an antihyperglycaemic lactate production by metformin arises from the splanchnic
agent used for the treatment of non-insulin-dependent dia- bed. In vivo and in vitro experiments in rats have been
betes mellitus (NIDDM) (Bailey, 1988; Bailey & Nattrass, undertaken to examine the effect of metformin on glucose
1988). The glucose-lowering action is associated with in- metabolism and lactate production by the intestine and liver.
creased peripheral glucose disposal, particularly by skeletal
muscle, and decreased hepatic glucose production (Bailey &
Puah, 1986; Prager et al., 1986; Nosadini et al., 1987; Wollen Methods
& Bailey, 1988). There is also evidence that metformin
decreases the rate of intestinal glucose absorption (Lorch, Animals
1971; Wilcock & Bailey, 1990a). The antihyperglycaemic
action of metformin requires the presence of insulin but the Adult male Wistar rats weighing about 200g were main-
drug does not stimulate insulin secretion (Bailey, 1988; Bailey tained in an air-conditioned room at 22 ± 2TC with 12 h light
& Nattrass, 1988). (08 h 00 min-20 h 00 min), and supplied a standard pellet
Concern about the use of metformin has focused on its diet (Rat breeding diet, Heygate and Sons, Northampton)
propensity to raise circulating lactate concentrations, mainly and tap water.
after meals, although the magnitude of this effect is generally
small (<2mmoll-') (Campbell et al., 1987; Jackson et al., Blood sampling studies
1987; Bailey, 1988; Bailey & Nattrass, 1988).
Metformin does not cause lactic acidosis if appropriately Rats were anaesthetized with sodium pentobarbitone (60 mg
prescribed, i.e. if patients with renal and hepatic insufficiency kg-' i.p.) and maintained under anaesthesia with further
are excluded (Bailey, 1988; Bailey & Nattrass, 1988; Camp- doses of 15 mg kg-' h- '. Rectal temperature was held at
bell, 1990). Extra lactate production during metformin ther- 34-36°C. The abdomen was opened and blood samples
apy was presumed to arise from peripheral tissues such as (50 ,A) were taken through fine heparinised needles inserted
skeletal muscle, but recent studies failed to confirm that at 4 sites: aorta (A) immediately anterior to the branching of
notion (Bailey & Puah, 1986; Jackson et al., 1987). the iliac arteries; inferior vena cava (IVC) adjacent to the
The present study investigates the possibility that increased right ilio-lumbar vein; hepatic portal vein (HPV) immediately
before branching into the liver; and hepatic vein (HV) as
close to the liver as possible. Test substances were admini-
stered by injection into the second loop of the jejunum and
Author for correspondence. massaged distally along the intestine. Groups of 12 h-fasted
1010 C.J. BAILEY et al.

rats received either saline (0.9% NaCi, 5 ml kg-') or metfor- infusion step of the hepatocyte isolation. Cell viability assess-
min hydrochloride (50 and 250 mg kg-' S ml'). Groups of ed by 0.1% trypan blue exclusion was accepted at >90%,
4 h fasted rats received either glucose (2 g kg-' 5 ml-') or and the number of viable cells was determined. Test incuba-
glucose with metformin hydrochloride (doses as above). Blood tions were performed with a suspension of 7 x 106 viable cells
samples were taken from all sites immediately before and at ml-' in a final volume of 1.5 ml. Test buffer was the same
60 and 120 min after administration of test substances. as for preincubation with the addition of 1.0 ptCi ml-'
Plasma glucose (Stevens, 1971) and lactate (Noll, 1974) were D-[U-'4C]-glucose, without and with insulin (10 8 mol I-1)
determined. and metformin (10-6-10-2 mol 1-'). Production of "'CO2
and lactate was measured as above after incubation for 2 h
Intestinal rings at 3T7C.
Fed rats were anaesthetized and treated by intrajejunal Chemicals
administration of either saline or metformin hydrochloride
(50 mg kg-') as above. After 2 h, rings of tissue (about Crystalline bovine insulin (24.3 iu mg-'), bovine serum albu-
30 mg) were prepared from the proximal, middle and distal min (fraction V, RIA grade), EGTA and collagenase (type
regions of the jejunum and ileum. The rings were washed in IV, from Clostridium histolyticum) were from Sigma Chemical
incubation buffer and incubated for 2 h at 37TC in 3 ml of Company, Poole, Dorset; D-[U-"'C]-glucose (specific activity
pregassed (95% 02:5% C02) Krebs Ringer bicarbonate (KRB) 270 mCi mmol-') was from Amersham International, Amer-
buffer, pH 7.4, containing bovine serum albumin 20 mg ml-', sham, UK; and pure metformin hydrochloride (batch 2452)
glucose 10 mmol 1', D-[U-'4C]-glucose 0.5 pCi ml-' and was from Lipha Pharmaceuticals, West Drayton.
insulin 10-8 mol 1-'. Production of 4CO2 and lactate was
determined (Bailey & Puah, 1986). Statistical analysis
Hepatocytes Data are presented as mean ± s.e.mean. Data were evaluated
for the effect of metformin by one-way analysis of variance
Hepatocytes were isolated from anaesthetized fed rats by a and differences between individual groups were compared by
modification of the collagenase method (Berry & Friend, Student's t test with Bonferroni's correction for multiple
1969). The following four buffers were infused at 5 ml min-' comparison. Differences were considered to be significant if
into the HPV without recirculation: calcium-free KRB supple- P<0.05.
mented with EGTA (0.5 mmol l') and sodium heparin (2
units ml-') for 5 min; calcium-free KRB for 5 min; KRB
supplemented with collagenase (0.5 mg min-') for 10 min; Results
and KRB supplemented with bovine serum albumin (20 mg
ml-') and glucose (10 mmol 1-) for 5 min. Buffers were Blood sampling studies
pH 7.4, saturated with 95% 02:5% CO2 and infused at 37C.
The liver was removed and cells were separated by disruption Intrajejunal administration of saline (5 ml kg-'), or metfor-
with dissecting needles. The suspension was filtered through min (50 and 250 mg kg-') to 12 h-fasted anaesthetized rats
muslin, washed and preincubated for 15 min at 37°C in did not significantly alter plasma glucose concentrations at
pregassed (95% 02:5% C02) buffer as used for the last each of the four sites sampled (IVC, HPV, HV and A) over

Metformin Metformin
Control 50 mg kg-' 250 mg kg-'
1U

If"

-5EE i
1~~~
0n A
¢ .A
0)

01

--I
0 60 120 0 60 120 0 60 120

O 2_ I
E
Ej
i
1.
C-
UE 1 - $ -- --II- _
Eco
FL 0 60 120 0 60 120 0 60 120
Time (min)
Figure 1 Plasma glucose and lactate concentrations of 12 h-fasted anaesthetized rats after intrajejunal administration of saline, and
50 and 250 mg kg-' metformin. (-) Inferior vena cava; (A) hepatic portal vein; (O) hepatic vein; (-) aorta. Values are mean with
s.e.mean shown by vertical lines, n = 6.
 METFORMIN AND SPLANCHNIC GLUCOSE METABOLISM 1011

Metformin Metformin
25 Control 50 mg kg-1 250 mg kg-'

20

-6E 15
E
a)
4T -"-==2
7
0D
Cen
0
.m
101-
E
0- i
5

QL L- J L __j L
0 60 120 0 60 120 o 60 120

3r I
.5
E 2
E A
0)
-W Y 0
cm0

E
Co
'4IS
0L 0 60 120 0 60 120 0 60 120
Time (min)
Figure 2 Plasma glucose and lactate concentrations of 4 h-fasted anaesthetized rats after intrajejunal administration of glucose
(2 g kg-') without and with 50 and 250 mg kg-' metformin. (M) Inferior vena cava; (A) hepatic portal vein; ('*) hepatic vein; (-)
aorta. Values are mean with s.e.mean shown by vertical lines, n = 10 for controls and n = 5 for the metformin-treated groups.

the 120 min duration of the study (Figure 1). Plasma lactate All values were significantly (P <0.05) greater than glucose
concentrations at each of the four sites were not significantly alone or glucose with 50 mg kg- ' metformin. It is noteworthy
altered by administration of saline or metformin, 50 mg kg-'. that 250 mg kg-' metformin caused a protracted increase in
However, 250 mg kg-' metformin increased plasma lactate plasma lactate in the HV of glucose-loaded rats.
concentrations (ANOVA, P < 0.05), with the greatest in-
crease occurring in the HPV (by 104%, P< 0.01) at 120 min.
Intrajejunal administration of glucose (2 g kg-') increased Intestinal rings
plasma glucose concentrations at all four sites sampled
(Figure 2). The greatest increase occurred in the HPV and Two hours after intrajejunal administration of 50 mg kg-'
the smallest increase in IVC as expected. Each dose of met- metformin, rings of proximal, middle and distal regions of
formin exerted an antihyperglycaemic effect but the pattern the jejunum and ileum were prepared and incubated in vitro
of the glucose response was different. Administration of with 10 mmol 1' U-['4C]-glucose. Glucose oxidation assessed
50 mg kg-' metformin with the glucose, reduced plasma by production of '4CO2, was decreased (by 19-38%) in the
glucose concentrations at 120 min in HPV (by 38%, P<0.01) mid-jejunum through to the mid-ileum (Figure 3). Lactate
and IVC (by 17%, P<0.05). When 250mg kg' metformin production was increased (by 21-25%) in the midjejunum
was administered with the glucose, plasma glucose concentra-
through to the proximal ileum. Since intestinal glucose meta-
tions were reduced at 60 min at all four sites (IVC by 21%, bolism was mainly anaerobic, metformin increased glucose
HPV by 37%, HV by 36% and A by 17%, all P<0.05), and utilization by 23% in the mid-jejunum to proximal ileum,
at 120 min in HPV (by 29%, P< 0.05). Plasma lactate con- and by 9.5% for the overall jejunum and ileum.
centrations were raised at all four sites at 60 min after intra-
jejunal glucose administration (IVC by 46%, HPV by 77%, Hepatocytes
HV by 270% and A by 121%, all P<0.05 compared with
time zero). In glucose-loaded rats, metformin increased lac- Glucose oxidation and lactate production were assessed using
tate concentrations in a dose-dependent manner. Administra- hepatocytes of fed rats incubated with metformin (10-6, 10-4
tion of 50 mg kg-' metformin with glucose produced greater and 10-2 mol 1-'). Metformin 10-6 and 10-4 moll did not '

increases in plasma lactate than glucose alone at all four sites significantly alter glucose oxidation or lactate production,
at 60 min (IVC by 48%, HPV by 52%, HV by 58% and A either in the absence or presence of 10-8 mol - insulin, 1

by 113% greater than glucose only, all P<0.05). Adminis- although 10-4mol 1' metformin increased mean values for
tration of 250 mg kg-' metformin with the glucose increased lactate production (Figure 4). Metformin, 102 mol 1', de-
plasma lactate at all four sites at 60 and 120 min: at 60 min creased glucose oxidation (about 30%) and increased lactate
IVC by 99%, HPV by 123%, HV by 62% and A by 118% production (about 60%) in the absence and presence of
greater than glucose only; at 120 min IVC by 103%, HPV by 10-8 mol 1-' insulin. Insulin (10-8 mol l-) alone did not
89%, HV by 160% and A by 99% greater than glucose only. significantly alter glucose oxidation or lactate production.
1012 C.J. BAILEY et al.

Jejunum Ileum However, the effect of metformin on net lactate production

Prox Mid Dist Prox Mid Dist
was greater during a glucose challenge, although the absolute
1000r magnitude of the increase in lactate concentrations was
always small (maximum increase 2.5 fold in the HPV).
c -
In the basal state lactate concentrations were consistently
._
higher in the HPV than HV, suggesting net extraction of
,3 _
lactate by the liver, which would buffer the extent of change
-0I
0 0) 500 - in peripheral lactate concentrations. During a glucose
CL -- challenge the concentration of lactate in the HV may exceed
0 EC
N1
that in the HPV, as evident at 120 min after 250 mg kg-'
u -
metformin. This is consistent with other evidence that when
OL
glucose and lactate concentrations are raised in the HPV, the
liver is no longer able to operate as a net lactate extractor
(Jackson et al., 1990). This would explain the clinical obser-
100r vation that metformin increases peripheral lactate concentra-
c tions mainly during meal absorption (Bailey & Nattrass,
.20 -
,

0i 50j Metformin (M) 0 10-6 lo-4 1o-2

Insulin (10-8 M) _ + - + - + - +

-
-i

01
Figure 3 Glucose oxidation to CO2 and lactate production by intes- 6000 -

tinal rings prepared from rats 2 h after intrajejunal administration of

50 mg kg-' metformin. Open columns indicate controls; stippled col-
umns indicate metformin-treated. Values are mean with s.e.mean CD
shown by vertical lines, n = 6. *P < 0.05 versus control (Student's t
test). Co

0
r-
0

E
Discussion -S
C

Clinically, metformin is prescribed in doses of 500-1000 mg 0

C_
twice or thrice daily with meals. Thus 50 mg kg-' metformin
given to a rat is equivalent on a weight related basis to the x
0
maximum clinical daily dose (3 g 60 kg- ') delivered as a 8)
Co
single bolus. In man, consumption of 1 g metformin produces 0

peak peripheral plasma concentrations of 1 5 x I0-5 mol 1 - '

at 1-2 h (Pentikainen et al., 1979; Tucker et al., 1981). In

rats, an oral bolus of 50 mg kg-' metformin produced similar
maximum concentrations of metformin in peripheral plasma
after 1 h (Wilcock et al., 1991). Since rats are less sensitive to
the antihyperglycaemic effect of metformin than man (Sterne, 0 L
1969), the present selection of metformin doses (50 and
250 mg kg-') and concentrations (10-6_ 10-2 mol 1- 1) was 6C 7
considered appropriate to investigate therapeutic and supra-
therapeutic effects.
Unlike sulphonylureas, metformin is not able alone to .C

cause clinical hypoglycaemia, and it has little effect on basal Co4

glucose concentrations in the non-diabetic state (Bailey, 1988; C.)

Bailey & Nattrass, 1988; Campbell, 1990). However, metfor- co

0
min lowers glucose concentrations in NIDDM patients and V-
0

in the glucose-loaded non-diabetic state, hence its designation 0

as an 'antihyperglycaemic' agent. Accordingly, metformin Ei

lowered the plasma glucose response to an intrajejunal glu- 30
cose challenge in the present study. Metformin 250 mg kg'
most strongly reduced HPV glucose concentrations consistent 0

with decreased intestinal glucose absorption (Lorch, 1971; 0

0)
Wilcock & Bailey, 1990a). Recent studies have shown that Q
metformin increases glucose utilization by the intestine (Peni-
caud et al., 1989; Wilcock & Bailey, 1990b), and our pre- J

liminary in vitro data indicated a concomitant increase in

lactate production (Wilcock & Bailey, 1990b). Although the
measurement of lactate concentrations at different sites does
not provide information on lactate turnover, the present 0 -

observation that 250 mg kg-l metformin produced the high- Figure 4 Glucose oxidation to CO2 and lactate production by rat
est lactate concentrations in the HPV, in both the basal and hepatocytes incubated for I h with metformin (10-6- 10-2 mol 1- ) in
glucose-loaded state, substantiates the theory that metformin the absence and presence of insulin (I0-I mol I-l). Values are mean
can increase intestinal net lactate production independently with s.e.mean shown by vertical lines, n = 8. *P <0.05 versus control
of intestinal glucose absorption (Wilcock & Bailey, 1990b). receiving same amount of insulin (Student's t test).
 METFORMIN AND SPLANCHNIC GLUCOSE METABOLISM 1013

1988; Campbell, 1990), and is consistent with a recent clinical production by hepatocytes is comparable with the effect of
study showing that a net increase in lactate production after the drug on the small intestine, but contrasts with the effect
metformin does not come from the periphery (Jackson et al., of lower (therapeutic) concentrations of metformin that
1987). Indeed in vitro studies have discounted muscle, fat, enhance glucose oxidation by peripheral insulin-sensitive
brain and skin as sources of extra lactate production by tissues, especially in mildly diabetic states (Frayn & Adnitt,
metformin (Wilcock & Bailey, 1990b). The present study 1972; Bailey & Puah, 1986; Wilcock & Bailey, 1990b).
excludes the periphery as the net source of the extra lactate Interestingly, rats appear to be more sensitive than mice to
because lactate concentrations were slightly higher in the the effects of metformin on glucose metabolism in the intes-
aorta than IVC. tine and liver; indeed even 10-2 mol 1-' metformin did not
Although therapeutic concentrations (10-6-10-4moll1') significantly alter glucose oxidation by these tissues from
of metformin did not significantly alter glucose oxidation or mice (Wilcock & Bailey, 1990b). In conclusion, the present
lactate production by hepatocytes, 10- mol 1' metformin study has provided evidence compatible with the view that
consistently increased mean values for lactate production. the main source of extra lactate produced by metformin is
Moreover, a very high concentration of metformin (10-2 mol the intestine. This is relatively small and may be extracted by
1-1) reduced glucose oxidation and increased lactate produc- the liver unless the liver is presented with high concentrations
tion by hepatocytes, consistent with the measurement of of both lactate and glucose.
highest glucose and lactate concentrations in the HV at
120 min after treatment of glucose-loaded rats with 250 mg The authors gratefully acknowledge the expert help and advice of Dr
kg-' metformin. The effect of a high metformin concentra- M.S. Billingham, Mr W. Cammes, Ms Heather Bull, Ms Susan L.
tion in reducing glucose oxidation and increasing lactate Turner and the Aston Animal Care Unit.

References
BAILEY, C.J. (1988). Metformin revisited: its actions and indications PENICAUD, L., HITIER, Y., FERRE, P. & GIRARD, J. (1989). Hypo-
for use. Diabet. Med., 5, 315-320. glycaemic effect of metformin in genetically obese (fa/fa) rats
BAILEY, C.J. & NATTRASS, M. (1988). Metformin. Balliere's Clinical results from an increased utilization of blood glucose by intestine.
Endocrinology and Metabolism, 2, 455-476. Biochem. J., 262, 881-885.
BAILEY, C.J. & PUAH, J.A. (1986). Effect of metformin on glucose PENTIKAINEN, P.J., NEUIVONEN, P.J. & PENTTILA, A. (1979). Phar-
metabolism in mouse soleus muscle. Diabet. Metab., 12, 212-218. macokinetics of metformin after intravenous and oral administra-
BERRY, M.N. & FRIEND, D.S. (1969). High yield preparation of tion to man. Eur. J. Clin. Pharmacol., 16, 195-202.
isolated rat liver parenchymal cells. J. Cell. Biol., 43, 506-520. PRAGER, R., SCHERNTHANER, G. & GRAF, H. (1986). Effect of
CAMPBELL, I.W. (1990). Sulphonylureas and metformin: efficacy and metformin on peripheral insulin sensitivity in non insulin depen-
inadequacy. In New Antidiabetic Drugs, ed. Bailey, C.J. & Flatt, dent diabetes mellitus. Diabet. Metab., 12, 346-350.
P.R. pp. 33-51. London: Smith-Gordon. STERNE, J. (1969). Pharmacology and mode of action of hypo-
CAMPBELL, I.W., DUNCAN, C.D., PATTON, N.W., BROADHEAD, T., glycaemic guanidine derivatives. In Oral Hypoglycaemic Agents
TUCKER, G.T. & WOODS, H.F. (1987). The effect of metformin on ed. Campbell, G.D., pp. 193-245. London: Academic Press.
glycaemic control, intermediary metabolism and blood pressure STEVENS, J.F. (1971). Determination of glucose by an automatic
in non-insulin-dependent diabetes mellitus. Diabet. Med., 4, analyser. Clin. Chim. Acta, 32, 199-201.
337-341. TUCKER, G.T., CASEY, C., PHILLIPS, P.J., CONNOR, H., WARD, J.D.
FRAYN, K.N. & ADNITT, P.I. (1972). Effects of metformin on glucose & WOODS, H.F. (1981). Metformin kinetics in healthy subjects
uptake by isolated diaphragm from normal and diabetic rats. and in patients with diabetes mellitus. Br. J. Clin. Pharmacol., 12,
Biochem. Pharmacol., 21, 3153-3162. 235-246.
JACKSON, R.A., HAWA, M.I., JASPAN, J.B., SIM, B.M., DISILVIO, L., WILCOCK, C. & BAILEY, C.J. (1990a). Reconsideration of inhibitory
FEATHERBE, D. & KURTZ, A.B. (1987). Mechanism of metformin effect of metformin on intestinal glucose absorption. J. Pharm.
action in non-insulin-dependent diabetes. Diabetes, 36, 632-640. Pharmacol., 43, 120-121.
JACKSON, R.A., SIM, B.M., HAWA, M.I. & DISILVIO, L. (1990). Effect WILCOCK, C. & BAILEY, C.J. (1990b). Sites of metformin-stimulated
of metformin on lactate turnover in non-insulin-dependent dia- glucose metabolism. Biochem. Pharmacol., 39, 1831-1834.
betes. Diabetologia, 33, A52. WILCOCK, C., WYRE, N.D. & BAILEY, C.J. (1991). Subcellular distri-
LORCH, E. (1971). Inhibition of intestinal absorption and improve- bution of metformin in rat liver. J. Pharm. Pharmacol., 43,
ment of oral glucose tolerance by biguanides in the normal and in 442-444.
the streptozotocin diabetic rat. Diabetologia, 7, 195-203. WOLLEN, N. & BAILEY, C.J. (1988). Inhibition of hepatic gluco-
NOLL, F. (1974). L( + ) lactate determination by LDH, GPT and neogenesis by metformin: synergism with insulin. Biochem.
NAD. In Methods of Enzymatic Analysis, 2nd edn. ed. Berg- Pharmacol., 37, 4353-4358.
meyer, H.U. pp. 1475-1479. New York: Academic Press.
NOSADINI, R., AVOGARO, A., TREVISAN, R., VALERIO, A., TES- (Received December 12, 1991
SARI, P., DUNER, E., TIENGO, A., VELUSSI, M., DEL PRATO, S., Accepted January 8, 1991)
DE KREUTZENBERG, S., MUGGEO, M. & CREPALDI, G. (1987).
Effect of metformin on insulin-stimulated glucose turnover and
insulin binding to receptors in type II diabetes. Diabetes Care, 10,
62-67.