Pharmacological Research Communications, Voi. 9, .No. l,~ 1977
RESPONSE OF "DIABETIC" MICE
Terence T. Yen, Mark M. Greenberg, Jean A. Allan and June M. Act0n The Lilly Research Laboratories, and Company,
Received5 April 1976
SUMMARY The compound p-fluorophenethylbiguanide glucose and plasma insulin concentrations mice, C57BL/KsJ-db/db.
lowered the blood
of genetic diabetic
The compound also depressed the respira-
tion of the db/db mice and caused the accumulation of plasma lactic acid.
Since these mice did not respond to the sulfonyl-
urea type of hypoglycemic compounds, differentiating
they are thus useful in those
the two types of hypoglycemic agents:
that stimulate insulin secretion and those that do not. INTRODUCTION The mutation diabetes
(db) was discovered in an inbred
mouse strain C57BL/KsJ at Jackson Laboratory and Coleman, by obesity,
for db are characterized
hyperglycemia and transitory hyperinsulinemia
(Coleman and Hummel, 1967).
Many studies have been conducted
on db/db mice in the past nine years
Pharmacological Research,Communications, Vol. 9, No. I j, 1977
very little is known about the response of the db/db mice to various pharmacological
especially the hypoglycemic I
This report describes
the effects of p-fluorophenethyl-
in db/db mice and.suggests
would be useful in discovering
that these mice
agents that do
not depend upon the stimulation of insulin secretion as the primary mode of action. We chose to use p-F-PEBG
instead of phenethylbiguanide
(PEBG) because the latter is first p-hydroxylated jugated to glucuronic p-Hydroxy-PEBG rats
acid in rats
(Murphy and Wick,
is not a very active pharmacological
(Cook, Blair, Gilfillan and Lardy,
and then con-
1968). agent in
and in mice
MATERIALS AND METHODS Male C57BL/KsJ-db/db mice were supplied by the Jackson Laboratory,
Bar Harbor, Maine.
Chow and water ad libitum. at about 25°C.
They were fed Purina Laboratory
Ambient temperature was maintained
Lighting was controlled by an automatic timer
to provide light from 6 a.m. to 6 p.m. For blood glucose determinations, tail with heparinized capillary tubes
blood was taken from the (Trident).
filtrates of blood were assayed with Worthington's using dextrose
as. the standard.
Plasma insulin concentrations the method of Wright et al
were determined according
(1968) using single-component
(Lilly) as the standard.
prepared by Dr. M. Bhatti and Mr. T. E. Cooper of our laboratories.
Guinea pig anti-porcine
from Miles Laboratories.
insulin serum was purchased
Pharmacological Research Colnmunications, Vol. 9, No. I, 1977
Respiration was measured in an apparatus described by McMahon et al
Since the animal chamber was designed to
accommodate a rat, three db/db mice were placed in the chamber to occupy the space and their respiration was monitored at the same time.
England Nuclear) Nuclear)
was administered, i.p.,
hours after p-F-PEBG
at 1 ~Ci/100 g body weight 3 i.p.) was given.
of respiration was started as soon as the *~C-tracer was injected, and lasted for at least an hour. Plasma lactic acid concentration was determined on perchloric acid filtrates with Calbiochem Rapid Lactat~ Reagents. lactate
was used as the standard.
The sources of various hypoglycemic compounds used in this study were:
Glicodiazine and Glisoxepide Glipizide O'Connell,
(Dr. N. Belcher,
(Dr. J. Mills, (Dr. J. S. Ward,
(Dr. K. Zellerhoff,
(Dr. P. W.
In all experiments unless otherwise specified, 2% Emulphor EL-620
(General Aniline and Film)
a mixture of
and saline was
used as a vehicle to dissolve or suspend the above hypoglycemic compounds.
Emulphor EL-620 is a polyoxyethylated vegetable oil.
The effect of p-F-PEBG on the blood glucose
levels of db/db mice over a period of 7 hours is shown in Figure i.
Given 50 mg/kg,
i.p. of p-F-PEBG,
the blood glucose concen-
tration of db___/dbmice decreased at 1 hour after dosing although
Pharmacological Research Communications, !/ol. 9, No, 1, 1977
was not significant.
ficant t h r o u g h
the 6th hour.
at 2 hours
o~m 4 0 II
LEGEND TO FIG.
The time course of the h y p o g l y c e m i c activity of p-fluorophenethylbiguanide (p-F-PEBG, 50 mg/kg, i.p.) in C 5 7 B L / K s J db/db mice (solid line) as compared to that of vehicle (2% ~u[phor-saline, dotted line). Both curves were compiled from data gathered from the same 4 groups of db/db mice, w i t h 8 m i c e in each group. Each group of m i c e was sampled before injection of p-F-PEBG. They were then sampled at 1 and 4 hours (group i); or 2 and 5 hours (group 2); or 3 and 6 hours (group 3); or 7 hours after injection. On a separate day, 2 weeks prior to the p-F-PEBG experiments, these same mice were injected with vehicle, 2% emulphor-saline, and bled correspondingly. The data points on both curves at 1 and 4 hours after injection, for example, w e r e all from group i. The "S" indicates that the v a l u e is s i g n i f i c a n t l y d i f f e r e n t from the "before injection" blood glucose level of that day at p<0.025. The change of b l o o d g l u c o s e level at 4 and 5 hours after v e h i c l e p r e s u m a b l y reflects a diurnal rhythm. M~ce were 2-1/2 to 3 months of age at the time of control experlments and 3 to 3-1/2 months old at the time of p-F-PEBG experiments. Blood glucose c o n c e n t r a t i o n s were d e t e r m i n e d on d e p r o t e i n i z e d filtrates of 25 ~i of h e p a r i n i z e d . w h o l e blood with W o r t h i n g t o n ' s glucostat.
Pharmacological Research Communications, .Vol. 9, No. 1, 1977 on the 7th hour. vehicle
The db/db mice given the same amount of
(0.i m l / 2 O g
i.p. of 2% Emulphor-saline)
showed a slight reduction in blood glucose level at 4 and 5 hours.
These decreases were not due to the injection o f vehicle
but due to a diurnal rhythm
p-F-PEBG was also active orally at 60 mg/kg suggesting that the comPound w a s w e l l doses,
(Table l) Atsimilar
i.p. or p.o., p-F-PEBG lowered the blood glucose level
of C57BL/KsJ-normal mice fed ad lib.
(data not shown).
compound was not active in db/db mice at 25 mg/kg,
Hours after p-F-PEBG
i.p. or p.o.
Oral activity of p-fluorophenethylbiguanide (p-F-PEBG) on blood glucose of db/db mice I Blood glucose (mean +- s.e. in mg %)
% of 0 time (mean +- s.e.)
p(vs 0 time) 2 ..
302.6 + 23.4
234.7 +- 26.9
75.0 +- 4.5
219.3 -+ 26.4
70.2 +- 4.7
IThe data presented in this table were compiled from three independent experiments using a total of 23 db/db mice with ages ranging from 3-1/2 months to 5-1/2 months. Each mouse was bled from the tail before p-F-PEBG and 3 and 5 hours after p-F-PEBG (60 mg/kg, p.o.). The compound was dissolved in 2% emulphorsaline. Blood glucose concentrations were determined on deproteinized filtrates from 25 ul of heparinized whole blood with Worthington's Glucostat. ZBased on Student's paired t-test.
In contrast to p-F-PEBG,
the following sulfonylureas did
not decrease the blood glucose level of d b / d b mice fed ad lib. at doses that were active in fasted normal mice: and tolbutamide at 150 mg/kg,
p.o., and Glicodiazine,
Glisoxepide and Glyburide at 50 mg/kg, p.o.
Pharmacological Research Communications, Vol. 9, No. 1, 1977
At 25 mg/kg,
i.p. or 50 mg/kg,
p-F-PEBG decreased the plasma insulin concentration of d b/db mice which were hyperinsulinemic
to begin with
result from the direct suppression of insulin secretion from pancreatic islets by PEBG as shown by Schatz et al Alternatively,
the lowering of plasma insulin level may indicate
a return of insulin sensitivity triggered by the reduction in blood glucose level.
Plasma insulin level of db/db mice after p - f l u o r o p h e n e t h y l b i g u a n i ~ (p-F-PEBG) I
0 TiRe 3 Hours (mean + s.e. in ~units/ml)
781.2 ± 103.2
615.0 ± 55.2
988.8 ± 122.7
278.4 ± 61.6
788.6 + 138.8
58.8 ± 38.2
IEach mouse was bled before p-F-PEBG and 3 hours after p-F-PEBG. Blood was collected in heparinized capillary tubes. Ten ~i of plasma were assayed according to the procedure of Wright et al (1968). Mice were 2-1/2 to 3 months old at the time of the experiments, p-F-PEBG was dissolved in water or saline. ZStudent's paired t-test was used to compare the levels at 0 time and 3 hours. 3Water
(0.1 mi/20 g, i.p.) was injected.
Respiration in vivo. m
The recovery of l~CO 2 from three
db/db mice during the first hour immediately after uniformly labeled Z~C-glucose or 1~C-fructose was administered was much less in the p-F-PEBG the tracer)
i.p., given 3 hours prior to
experiment than in the vehicle experiment.
l~C-glucose as a tracer, vehicle-treated mice expired 18.5% of the radioactivity
injected during that hour whereas the same
Pharmacological Research Communications, Vol. 9, No. 1, !977
three mice treated w i t h p-F-PEBG expired only 6.6% of the radiop
For * ~ C - f r u c t o s e ,
expired 18.6% of t h e radioactivity given whereas t h e same mice treated with p-F-PEBG expired only 8.8% of the radioactivity injected.
However, for both 19C-glucose and l~C-frL~ctose, the
specific activitY of the CO z expired (I~CO2/C02) did ~.ot change because of the p-F-PEBG treatment.
This indicated a depression
of respiration in general instead of a specific depression of the Oxidation of glucose or fructose.
D e p r e s s i o n of oxidation
of various substrates in different tissues by PEBG in vitro has been demonstrated and suggested by several groups of workers as a result of PEBG inhibition of oxidative, phosphorylation (S~ling et al, 1967, pereira, Jangaard and Pinson, Pereira and Pinson, 1968, Elkeles,
Our in vivo data
support this hypothesis. Plasma lactic acid.
The characteristic accumulation of
lactic acid in plasma caused by PEBG
(Assan, 1975) can also be
demonstrated with p-F-PEBG in both young and mature db/db mice (Table 3) .
The data reported here demonstrate unequivocally the effect of p-F-PEBG on db/db mice.
Administration of p-F-PEBG to d b/db mice
effectively lowers the blood glucose and plasma insulin concentrations, depresses respiration and produces a rise in plasma lactic acid level.
These pharmacological effects are characteristic for
biguanide s .
Similar effects have been observed in humans with
Our data on db/db mice thus support the n o t i o n that bigua-
n/des exert their hypoglycemic effects through alteration of
Pharmacological Research Communications, Vol. 9, No. 1, 1977
46 T a b l e 3. m,
Plasma lactic acid level of db/db mice after p-fluorophenethylbiguanide (p-F-PEBG)*
3-1/2 - 4-1/2 ,
i-1/2 _ _
p F PEBG2
s . e . in mg %)
, • ,
39.4 +_ 4.1 31.9 -+ 3.9
(N=6) I 54.6 + (N=I2) . m
83.5 + i0.9
IMice were bled by decapitation or from the tail 3 hours after dosing. Blood was collected in heparinized tubes. A perchloric acid filtrate was prepared from 20 ~I or 50 ~1 plasma. Lactic acid concentration was determined with Calbiochem Rapid Lactate Reagents using lithium lactate as the standard. 2p-F-PEBG was dissolved in 2% Emulphor-water and administered at 75 mg/kg, i.p. (15 mg/ml). Mice serving as controls were dosed with the same volume of vehicle (0.i ml/20 g body weight). 3Probability was determined by Student's t-test comparing p-F-PEBGtreated db/db mice with vehicle-treated db/d b mice.
cellular oxidative processes,
possibly increasing anaerobic glyco-
lysis by inhibiting aerobic metabolism.
None of the six sulfonylureas tested affected the blood glucose level of db/db mice. tant and sulfonylureas least acutely,
Since db/db mice are insulin resis-
are known to promote insulin secretion,
the lack of response to sulfonylureas
is not a sur-
prise. The fact that db/db mice respond to biguanides and do not respond to sulfonylureas
prompts us to propose that these mice be
used in searching for new hypoglycemic agents.
in such a system would have peripheral mechanisms and would not enhance llpogenesis should be therapeutically
as insulin does.
like biguanides Such agents
useful for both juvenile type and
maturity-onset diabetes. It should also be possible,
using db/db mice,
to find hypo-
glycemic agents llke.biguanides but devoid of their lactic acid-
Such agents would offer a great improvement
Pharmacological Research Communications, Vol. 9, No. 1, 1977 in diabetes
therapy by possibly acting by mechanisms distinct and
different from those of PEBG and p-F-PEBG. Studying effects of various hypoglycemic
agents such as
p-F-PEBG in db/db mice, on the other hand, will also lead to a better understanding
of the etiology of diabetes
an understanding which may be applicable
in these mice,
to certain types of human
ACKNOWLEDGEMENTS The authors are grateful to Dr. N. Belcher (Pfizer), Dr', J. Mills (Lilly), Dr. P. W. O'Connel (Upjohn), Dr. J. S. Ward (Lilly) and Dr. K. Zellerhoff (Bayer) for their generous supply of various hypoglycemic compounds used in this study. The authors are also indebted to Dr. R. J. Hosley (Lilly) for his assistance in obtaining some of these compounds.
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Hummel, K. P., Dickie, M. M., and Coleman, D. L. : Sci. 153, 1127 (1966) Jangaard, N. O., Pereira, J. N., and Pinson, R.: Diabetes 17,
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