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Effect of DG5128 on epinephrine and glucagon induced glucose output from the isolated perfused rat liver
Life Sciences, Vol. 42, pp. 129-136 Printed in the U.S.A.
Pergamon Journals J
EFFECT OF DG5128 ON EPINEPHRINE AND GLUCAGON INDUCED GLUCOSE OUTPUT FROM THE ISOLATED PERFUSED RAT LIVER Masaru Usami I)3) Yutaka Seino 1), Tomohiko T a m i ~ t o Michiyo Seno 2), G y o ~ n Koh 2), M a s a f u m ~ O h n o ~j, Masaki Ikeda ~j and Hiroo Imura ~j
1) '
l)Di~$sion, of Metabolism and Clinical Nutrition and ~JSecond Division, Department of Medicine, Ky@~o University School of Medicine, Kyoto 606, Japan oJThe Diabetes Center, Ikeda Hospital, Amagasaki, Hyogo 661, Japan (Received in final form November 9, 1987) Summary The effect of a specific ~p-adrenergic antagonist 2-[2(4,5-dihydro-l.H-imidazol-2-yl)-l-phenyl-ethyl] pyridine dihydrochloride sesquihydrate (DG5128), on the glucose output by epinephrine and/or glucagon was studied using the perfused rat liver. The administration of DG5128 alone did not affect the glucose output. However, DG5128 produced a significant inhibitio~ of the increased glucose output when induced by 10-UM epi~Nphrine alone or 10-UM epinephrine plus 1.4 x 10-±UM glucagon. There were no1~ignificant changes of the glucose output by 1.4 x 10-~VM or 7.0 x 10-~±M glucagon alone. On the other hand, addition of l ~ / m l insulin to the perfusate suppressed the 7.0 x 1 0 - ~ M g l u c a g o n - i n d u ~ d glucose output, but failed to decrease the 1.4 x 10-~VM glucagon e f f e c ~ DG5128 suppressed further the glucagon (7.0 x 10-±~M)-induced increase of glucose output in the presence of insulin. These results suggest that DG5128 produces a hypoglycemic effect partly through an inhibition of the increased hepatic glucose output elicited by epinephrine and glucagon. 2-[2-(4,5-dihydro-l.H-imidazol-2-yl)±l-phenyl-ethyl] pyridine dihydrochloride sesquihydrate (DG5128) is a newly synthetised highly specific ~2-adrenergic antagonist, which might have a role as a hypoglycemic agent (i) by stimulating the insulin release from the pancreatic islets or the isolated perfused pancreas by an effect on ~2-adrenergic receptors (2,3). The significance of the extrapancre~tic effect of hypoglycemic agents has been discussed (4,5) and several investigators have reported that hepatic carbohydrate metabolism has been influenced by the hypoglycemic agent (6,7,8). In the present study, we have demonstrated the effect of DG5128 on the activation of the glucose output elicited by epinephrine and glucagon from the perfused rat liver. 0024-3025/88 $3.00 + .00 Copyright (c) 1988 Pergamon Journals Ltd.
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~2~Blocker Effect on Glucose Output
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Materials and Methods Male wistar strain rats (200-210 g body weight) having free access to food were used for this experiment. The liver perfusion in situ was performed by the system described by M o r t i m o r e et al. (9) with minor modifications (I0). Preperfusion for 20 minutes was performed with a recirculation system. Afterwards, the perfusion was changed to a non-recirculation system and the sampling was begun. The p e r f u s a t e v o l u m e was 150 ml at a f l o w rate of i0 m l / m i n . The perfusate consisted of 20 % washed bovine erythrocyte and Krebs Ringer bicarbonate buffer containing 4 % bovine albumin. It was g a s s e d w i t h a h u m i d i f i e d m i x t u r e of 95 % 02 and 5 % CO 2 and pH was monitored using a glass electrode and maintained at 7.4 by titration with IN NaOH. The perfusate reservoir and oxygenator were water jacketed and thermostatically controlled at 37 C. In the first experiment, 10 -6 M epinephrine and/or 1.4 x 10-10M glucagon was injected into the perfusate. After 3 minutes, 2-[2-(4,5-dihydro-l.H-imidazol-2-yl)-l-phenyl~ethyl] pyridine dihydrochloride sesquihydrate (DG5128), which was synthesized in the biochemical laboratory of Daiichi Seiyaku Co., Ltd., (I) and provide generously, was added to the perfusate at 6 minutes after the injection and the perfusion was continued for the next 8 minutes. For controls, only glucagon and/or epinephrine was injected into the perfusate. In the second experiment, insulin (i mU/ml) was administrated into the perfusate 5 minutes before the sampling, with or without the addition of DG5128 ~ e n the sampl~ng was started. In both cases, glucagon (1.4 x 10-1UM, 7.0 x 10-1±M) was infused into the perfusate 5 minutes after the start of sampling and the perfusion was continued for the n e x ~ 0 minutes. ~ control experiment, only glucagon (1.4 x 10-~VM, 7.0 x 1 0 - ~ M ) was infused at the same condition as discribed above. Each I minutes samples of effluent was collected and the glucose concentration was measured by the glucose oxidase method. Glucose output (uM/g.liver) was calculated for each sample. The sum of c h a n g e s in the g l u c o s e o u t p u t for 8 m i n u t e s a f t e r the addition of DG5128 in the first experiment and for the I0 minutes in the second experiment, from the rate immediately before the administration, was calculated and expressed as integrated increments of the glucose output. The statistical analysis was performed by the analysis of valiance and the Student's "t" test. Results The first experiment: The basal rate of glucose output after the preperfusion was--6~-± 7 ~M/g.liver (mean ± SE) and the addition of DG5128 (0.I m M or I mM) did not produce any significan~ changes of the glucose output. The administration of 10-UM epinephrine into the perfusate elicited a significant increase in the glucose output. The basal rate of glucose output was 78 ± 14 ~M/g. liver and the level at 6 minutes after the administration of epinephrine was 187 ± 21 ~M/g.liver in controls, in which DG5128 was not a d d e d to p e r f u s a t e m e d i u m . The a d d i t i o n of 0.I m M D G 5 1 2 8
Vol. 42, No. 2, 1988
~2-Blocker Effect on Glucose Output
Epinephrine 10"6M
DG
!
9
c,----,o Control(N=5) - - DG 0.1rnM(N=5) ~----A DG lmM(N=6)
200
~ 100.
3
5
Fig.
min.
16
1
Effects of DG5128 (• • 0.i mM, A - - - - - A 1 mM) on the 1 0 - U M e p i n e p h r i n e - i n d u c e d rise in glucose output f r o m the p e r f u s e d rat liver. Values are m e a n s ± SE, w i t h the n u m b e r s of a n i m a l s given in parenthesis. * D i f f e r e n t f r o m control, P < 0.05.
GLuc(:lgon1.4x10-~M
4
o---.o Control(N=6) : - DG0.1ram(N=5) A-.--~ DG froM(N=5)
DG
1
200 d
== • 100. o
~(~. .~...~.'" ./
,,.D
Fig.
2
Effects o ~ n D G 5 1 2 8 (e • 0.I mM, A-A i mM) on the 1.4 × 1 0 - ~ U M g l u c a g o n - i n d u c e d rise in glucose output f r o m the p e r f u s e d rat liver. Values are m e a n s ± SE, w i t h the n u m b e r s of a n i m a l s given in parenthesis.
13]
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tended to inhibit the increased rate of the glucose output induced by epinephrine, compared to controls, but it was not significant (Fig. i). However, 1 m M DG5128 produced a significant decrease in the activated glucose output elicited by epinephrine. In addition, glucose output expressed as integrated increments from the rate of glucose output before the addition of DG5128 were -105 ± 30 and -339 ± 32 ~M/g.liver in the 0.I m M and 1 m M DG5128 administration, respectively. These values were significantly lower than controls (108 ± 65 ~M/g.liver; P < 0.05 vs 0.i m M DG5128, P < 0.01 vs 1 m M DG5128) as s h o w n F i g u r e 4. On the other hand, the addition of 1.4 × 10-10M glucagon produced an obvious increase of the glucose output. The basal rate of g l u c o s e o u t p u t and the v a l u e at 6 m i n u t e s a f t e r g l u c a g o n administration was 78 ± II and 172 ± 15 ~M/g.liver, respectively. The rate and changes of the glucose output after the addition of DG5128 (0.I mM, 1 mM) were not significantly different from controls as shown Figure 2 and 4.
Th e simultaneous administration of 10-6M epinephrine and 1.4 x lO-IUM glucagon increased glucose output from the basal rate of 64 ± I0 ~M/g.liver to the rate of 310 ± 40 ~M/g.liver at 6 minutes after the administration. And the addition of 0.I m M or 1 m M DG5128 produced a significant decrease of the increased glucose output, when induced by both epinephrine and glucagon, compared to controls (Fig. 3). Expressed as integrated changes of the glucose output after the addition of the agent, the values d u r i n g the a d m i n i s t r a t i o n of 0.i m M and 1 m M D G 5 1 2 8 w e r e 264 ± 99 and -114 ± 94 ~M/g.liver, respectively, significantly lower than the 625 ± 109 ~M/g.liver of controls (P < 0.05 vs 0.i m M DG5128, P < 0.01 vs 1 m M DG5128), as s h o w n F i g u r e 4.
Epinephrinel0-~M Glucogon 1.4x10~°M
o----o Control (N=5) : = DG 0.1mM(N=6) ~ - . 4 DG 1 r a M ( N = 6 ) DG
400
~s
~-~"-~"~
rain.
16
r:
E
\
o
300
200
lOO
1
3
5
9
Fig.
3
Effects of DG5128 (e • 0.I m M , = & - - - - - & i mM) on the rise of g ~ c o s e output by both 10-UM epinephrine and 1.4 x 10-±UM glucagon from the perfused rat liver. Values are means ± SE, with the numbers of animals given in parenthesis. *Different from Control, P < 0.01.
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~2-Blocker Effect on Glucose Output
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The second experiment: Insulin infusion ten~e~ to lower the increased glucose o-~put induced by 1.4 x 10-~VM glucagon, but it was not significantly different from the control values. In addition, the a d m i n i s t r a t i o n of DG5128 did not produce any significant changes in the increased glucose output by 1.4 x 10-~VM glucagon in the absence and presence of insulin. The administration of 7.0 x 10-11M glucagon produced al~Rst the same increase of the glucose output as that of 1.4 x 10-±UM glucagon, as shown Table I. In this study, however, the insulin infusion inhibited significantly t h ~ i n c r e a s e d rate of the glucose output induced by 7.0 x 10-~IM glucagon. Glucose output expressed as integrated increments from the rate of the glucose output before the addition of glucagon was 1683 ± 108 pM/g.liver in the presence of insulin, significantly lower than the value of 2064 ± Iii ~M/g.liver in control experiment. The addition of DG5128 in the absence of i n s ~ i n did not affect the glucose output elicited by 7.0 x 10-~IM glucagon, but decreased further the 7 . 0 x 10-±±M glucagon-induced increase of the glucose output when insulin was perfused (Table I).
~neph~
OOI
Glucogon
F~oine~rinepl~ C 4 ~
600 UM 4
2OO
T 0
~
[X;O.~rnMDGlrn~ Control OGIllr~ lY~ImM
-200
Con(rol
DG01rnM
Control~
Fig. 4 Changes in gluco#~ output induced by 10-6M epinephrine and/or 1.4 x 10 -I M glucagon after the administration of DG5128 in the perfused rat liver. Values are means ± SE. *,**Different from each control P < 0.05, P < 0.01
134
~2-Blocker Effect on Glucose Output
Table I.
Vol. 42, No. 2, 1988
Effects of DG5128 and/or insulin on glucagon-induced glucose output. Glucose output
(HM/g.liver)
Basal rate
Rate at 8 min
(N)
Net increase for I0 minutes
Glucagon 1.4 x 10-10M only + DG5128 + Insulin + DG5128 + Insulin
(5) (4) (4) (4)
87 85 73 93
± 7 ± 9 ± 12 ± i0
380 375 346 358
± ± ± ±
31 28 13 30
2174 2056 1839 1866
i 191 ± 125 ± 98 i 135
Glucagon ~ 7.0 x 10-±~M only + DG5128 + Insulin + DG5128 + Insulin
(5) (4) (4) (4)
62 72 70 72
± I0 ± I0 ± 19 ± 9
363 346 297 222
± ± ± ±
21 16 20* 36 *,+
2064 1823 1683 1080
± ± ± ±
iii 120 108" 133 **,+
Glucagon was added 5 min after the beginning of sampling as described in Materials and Methods. Insulin (i mU/ml) and D G 5 1 2 8 (I mM) w e r e a d d e d I0 m i n and 5 m i n b e f o r e the infusion of glucagon, respectively. Basal rate shows the level of glucose output before glucagon infusion and rate at 8 min indicates the level 8 min after glucagon infusion. Net increase in glucose output above the basal levels was c u m u l a t e d for a i0 m i n p e r i o d a f t e r the a d d i t i o n of glucagon. *P < 0.05, **P < 0.01 vs g l u c a g o n 7.0 x 1 0 - 1 1 M only +P < 0.05 vs glucagon + insulin Discussion Epinephrine and glucagon at physiological concentrations are well k n o w n to activate glucose production from the liver (11,12, 13). In the present study with the perfused rat liver, the increase in glucose production induced by these two hormones must be caused by glycogenolysis, since the liver perfusion was carried out in the absence of any gluconeogenic substrates. The increased rate of the glucose output is greater when both epinephrine and glucagon are administered together than when either hormone is administered alone. The present results are consistant with the work of Studer et al. using perfused hepatocytes (14). They found that the simultaneous administration of the two hormones caused an increase in phosphorylase activity which was quantitatively additive. In the present study, the administration of DG5128, an ~p-adrenergic antagonist, reduced the increased glucose output elicited by epinephrine alone or epinephrine plus glucagon. The effects of epinephrine on the carbohydrate m e t a b o l i s m in the liver are mediated by ~ and 6 adrenergic receptors. Rizza et al. (15) have reported that the stimulation of glucose production and the suppression of glucose clearance by epinephrine in man is predominantly the result of 6-
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~2-Blocker Effect on Glucose Output
135
adrenergic mechanisms. On the other hand, it has been investigated that the effects of epinephrine on the hepatic carbohydrate m e t a b o l i s m in rats are mediated predominantly by ~adrenergic receptors (10,16). The differences in species and experimental methods may account for the contradictory results. Recently, ~-adrenergic receptors have been subdivided into two types, ~i and ~p (17). Several investigators have suggested that the ~l-adrenerg~c action in the liver is mediated through an elevation of cytosolic Ca Z+ and that the ~2-adrenergic action is dependent on the inhibition of adenylate cyclase (18). However, Studer et al. (14) have reported that there is no evidence of an ~2-adrenergic inhibition of adenylate cyclase. These conflicting reports indicate that the mechanism of ~p-adrenergic action has not yet been clarified. Aggerback et alZ (19) have shown that prazocin and yohimbine, which are ~I- and ~p -adrenergic antagonists, respectively, produced an inhiSition of the glycogen phosphorylase stimulated by epinephrine in isolated rat hepatocytes. Therefore, it was suggested that the inhibition of the increased glucose output by DG5128 may be produced through a decrease of phosphorylase activity, as well as by yohimbine. On the o t h e r hand, it is well k n o w n that g l u c a g o n is important for the maintenance of the glucose level at the physiological condition (20), and this hormone plays an important role in the hyperglycemia of diabetes mellitus (21). In the present study, we have observed that glucose prp~uctions induced by @@ch of the two doses of glucagon (1.4 x 10-~UM and 7.0 x 1 0 - ~ M ) were almost the same. In addition, insulin infusion inhibits glucose production1~nduced by physiological concentration p~ glucagon (7.0 x 1 0 - ~ M ) , but not by higher dosage (1.4 x 10-±UM). These results have indicated that 7.0 x 10-±~M glucagon is sufficient for maximal glucose production by glucagon in this system and demonstrated further that insulin could not suppress the glucose production elicited by the higher concentration of glucagon (1.4 x 10-~UM). Kameda et al. have reported that DG5128 fails to decrease the elevated blood glucose in the streptozotocin-induced diabetic rats (2). The rats are regarded, however, as a model of insulindependent diabetes mellitus (IDDM), because the blood glucose levels in these rats are above 300-400 mg/dl, indicating that these rats are at the severe insulin deficient state. It is well known that the hypoglycemic agents such as sulfonylureas are ineffective for severe insulinopenic patients. Therefore, even if DG5128 does not decrease the blood glucose levels in these rats, we can not rule out the p o s s i b i l i t y of a h y p o g l y c e m i c effect of the agent. Furthermore, the present observation, in which DG5128 produces an inhibitory effect on the glucose output induced by physiological concentration of glucagon only in the presence of insulin, in addition to its stimulation of insulin secretion, leads to support the possibility that the substrate could be a hypoglycemic agent for the treatment of non-insulin dependent diabetes mellitus (NIDDM) like sulfonylureas. Acknowledgements We are grateful to Ms. H. Tachikawa preparation of this manuscript.
for aid in the
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~2-Blocker Effect on Glucose Output
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References i. 2.
. 4.
5. 6. 7. 8. 9. I0. ii 12 13 14. 15 16 17 18 19 20 21.
F. ISHIKAWA, Chem. Pharm. Bull. 28 1394-1402 (1980). K. K A M E D A , S. ONO, I. K O Y A M A and Y. ABIKO, A c t a Endocrinol. 99 410-415 (1982). G. KOH, Y. SEINO, K. TSUDA, S. NISHI, H. ISHIDA, J. TAKEDA, H. FUKUMOTO, T. TAMINATO and H. IMURA. Life Sci. 40 1113-1118 (1987). J.M. F E L D M A N , H.E. LEBOVITZ, Arch. Intern. Med. 123 314-320 (1969). J. ROTH, Ann. Intern. Med. 75 607-613 (1971) S.A. BLUMENTHAL and K.R. WHITMER, Diabetes 28 646-650 (1979). A. MATSUTANI, K. KAKU and T. KANEKO, Diabetes 33 495-498 (1984). T.B. PATEL, Am. J. Physiol., 250 E82-E86 (1986). G.E. MORTIMORE, F. TIENZE, D.W. STETTEN, Diabetes 8 307-314 (1959). Y. SEINO, S. SEINO, J. T A K E M U R A , K. TSUDA, S. NISHI H. ISHIDA, M° SENO, M. USAMI, M. IKEDA and H. IMURA, Endocrinology 114 457-461 (1984). N.J. HUTSON, F.T. B R U M L E Y , F.D. A S S I M A C O P O U L O S , S.C. H A R P E R and J.H. EXTON, J. Biol. Chem. 251 5200-5208 (1976). P.F. B L A C K M O R E , F.T. BRUMLEY, J.L. M A R K S and J.H. EXTON, J. Biol. Chem. 253 4851-4858 (1978). L. SACCA, R. S H E R W I N and P. FELIG, Am. J. Physiol. 236 EII3-EII7 (1979). R.K. STUDER, K.W. S N O W D O W N E and A.B. BORLE, J. Biol. Chem. 259 3596-3604 (1984). R.A. RIZZA, P.E. CRYER, M.W. H A Y M O N D and J.E. GERICH, J. Clin. Invest. 65 682-689 (1980). P.L.A. SHERLINE and W.H. GLINSMANN, Endocrinology 91 680690 (1972). S. BERTHELSON and W.A. PETTINGER, Life Sci. 21 595-606 (1977). J.A. G A R C I A - S A I N Z , B.B. H O F F M A N , S.Y. LI, R.J. L E F K O W I T Z and J.N. FAIN, Life Sci. 27 953-961 (1980). M. AGGERBECK, G. GUELLAEN and J. HANOUNE, Biochem. Pharma. 29 643-645 (1980). ~.E. L I L J E N Q U I S T , G.L. M U E L L E R , A.D. C H E R R I N G T O N , U. KELLER, J.L. CHIASSON, J.M. PERRY, W.W. LACY and D. RABINOWITZ, J. Clin. Invest. 59 369-374 (1977). J.D. BEST, R.G. J U D Z E W I T S C H , M.A. PFEIFER, J.C. BEARD, J.B. HALTER and D. PORTE JR., Diabetes 31 333-338 (1982).
Pergamon Journals J
EFFECT OF DG5128 ON EPINEPHRINE AND GLUCAGON INDUCED GLUCOSE OUTPUT FROM THE ISOLATED PERFUSED RAT LIVER Masaru Usami I)3) Yutaka Seino 1), Tomohiko T a m i ~ t o Michiyo Seno 2), G y o ~ n Koh 2), M a s a f u m ~ O h n o ~j, Masaki Ikeda ~j and Hiroo Imura ~j
1) '
l)Di~$sion, of Metabolism and Clinical Nutrition and ~JSecond Division, Department of Medicine, Ky@~o University School of Medicine, Kyoto 606, Japan oJThe Diabetes Center, Ikeda Hospital, Amagasaki, Hyogo 661, Japan (Received in final form November 9, 1987) Summary The effect of a specific ~p-adrenergic antagonist 2-[2(4,5-dihydro-l.H-imidazol-2-yl)-l-phenyl-ethyl] pyridine dihydrochloride sesquihydrate (DG5128), on the glucose output by epinephrine and/or glucagon was studied using the perfused rat liver. The administration of DG5128 alone did not affect the glucose output. However, DG5128 produced a significant inhibitio~ of the increased glucose output when induced by 10-UM epi~Nphrine alone or 10-UM epinephrine plus 1.4 x 10-±UM glucagon. There were no1~ignificant changes of the glucose output by 1.4 x 10-~VM or 7.0 x 10-~±M glucagon alone. On the other hand, addition of l ~ / m l insulin to the perfusate suppressed the 7.0 x 1 0 - ~ M g l u c a g o n - i n d u ~ d glucose output, but failed to decrease the 1.4 x 10-~VM glucagon e f f e c ~ DG5128 suppressed further the glucagon (7.0 x 10-±~M)-induced increase of glucose output in the presence of insulin. These results suggest that DG5128 produces a hypoglycemic effect partly through an inhibition of the increased hepatic glucose output elicited by epinephrine and glucagon. 2-[2-(4,5-dihydro-l.H-imidazol-2-yl)±l-phenyl-ethyl] pyridine dihydrochloride sesquihydrate (DG5128) is a newly synthetised highly specific ~2-adrenergic antagonist, which might have a role as a hypoglycemic agent (i) by stimulating the insulin release from the pancreatic islets or the isolated perfused pancreas by an effect on ~2-adrenergic receptors (2,3). The significance of the extrapancre~tic effect of hypoglycemic agents has been discussed (4,5) and several investigators have reported that hepatic carbohydrate metabolism has been influenced by the hypoglycemic agent (6,7,8). In the present study, we have demonstrated the effect of DG5128 on the activation of the glucose output elicited by epinephrine and glucagon from the perfused rat liver. 0024-3025/88 $3.00 + .00 Copyright (c) 1988 Pergamon Journals Ltd.
130
~2~Blocker Effect on Glucose Output
Vol. 42, No, 2, 1988
Materials and Methods Male wistar strain rats (200-210 g body weight) having free access to food were used for this experiment. The liver perfusion in situ was performed by the system described by M o r t i m o r e et al. (9) with minor modifications (I0). Preperfusion for 20 minutes was performed with a recirculation system. Afterwards, the perfusion was changed to a non-recirculation system and the sampling was begun. The p e r f u s a t e v o l u m e was 150 ml at a f l o w rate of i0 m l / m i n . The perfusate consisted of 20 % washed bovine erythrocyte and Krebs Ringer bicarbonate buffer containing 4 % bovine albumin. It was g a s s e d w i t h a h u m i d i f i e d m i x t u r e of 95 % 02 and 5 % CO 2 and pH was monitored using a glass electrode and maintained at 7.4 by titration with IN NaOH. The perfusate reservoir and oxygenator were water jacketed and thermostatically controlled at 37 C. In the first experiment, 10 -6 M epinephrine and/or 1.4 x 10-10M glucagon was injected into the perfusate. After 3 minutes, 2-[2-(4,5-dihydro-l.H-imidazol-2-yl)-l-phenyl~ethyl] pyridine dihydrochloride sesquihydrate (DG5128), which was synthesized in the biochemical laboratory of Daiichi Seiyaku Co., Ltd., (I) and provide generously, was added to the perfusate at 6 minutes after the injection and the perfusion was continued for the next 8 minutes. For controls, only glucagon and/or epinephrine was injected into the perfusate. In the second experiment, insulin (i mU/ml) was administrated into the perfusate 5 minutes before the sampling, with or without the addition of DG5128 ~ e n the sampl~ng was started. In both cases, glucagon (1.4 x 10-1UM, 7.0 x 10-1±M) was infused into the perfusate 5 minutes after the start of sampling and the perfusion was continued for the n e x ~ 0 minutes. ~ control experiment, only glucagon (1.4 x 10-~VM, 7.0 x 1 0 - ~ M ) was infused at the same condition as discribed above. Each I minutes samples of effluent was collected and the glucose concentration was measured by the glucose oxidase method. Glucose output (uM/g.liver) was calculated for each sample. The sum of c h a n g e s in the g l u c o s e o u t p u t for 8 m i n u t e s a f t e r the addition of DG5128 in the first experiment and for the I0 minutes in the second experiment, from the rate immediately before the administration, was calculated and expressed as integrated increments of the glucose output. The statistical analysis was performed by the analysis of valiance and the Student's "t" test. Results The first experiment: The basal rate of glucose output after the preperfusion was--6~-± 7 ~M/g.liver (mean ± SE) and the addition of DG5128 (0.I m M or I mM) did not produce any significan~ changes of the glucose output. The administration of 10-UM epinephrine into the perfusate elicited a significant increase in the glucose output. The basal rate of glucose output was 78 ± 14 ~M/g. liver and the level at 6 minutes after the administration of epinephrine was 187 ± 21 ~M/g.liver in controls, in which DG5128 was not a d d e d to p e r f u s a t e m e d i u m . The a d d i t i o n of 0.I m M D G 5 1 2 8
Vol. 42, No. 2, 1988
~2-Blocker Effect on Glucose Output
Epinephrine 10"6M
DG
!
9
c,----,o Control(N=5) - - DG 0.1rnM(N=5) ~----A DG lmM(N=6)
200
~ 100.
3
5
Fig.
min.
16
1
Effects of DG5128 (• • 0.i mM, A - - - - - A 1 mM) on the 1 0 - U M e p i n e p h r i n e - i n d u c e d rise in glucose output f r o m the p e r f u s e d rat liver. Values are m e a n s ± SE, w i t h the n u m b e r s of a n i m a l s given in parenthesis. * D i f f e r e n t f r o m control, P < 0.05.
GLuc(:lgon1.4x10-~M
4
o---.o Control(N=6) : - DG0.1ram(N=5) A-.--~ DG froM(N=5)
DG
1
200 d
== • 100. o
~(~. .~...~.'" ./
,,.D
Fig.
2
Effects o ~ n D G 5 1 2 8 (e • 0.I mM, A-A i mM) on the 1.4 × 1 0 - ~ U M g l u c a g o n - i n d u c e d rise in glucose output f r o m the p e r f u s e d rat liver. Values are m e a n s ± SE, w i t h the n u m b e r s of a n i m a l s given in parenthesis.
13]
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~2-Blocker Effect on Glucose Output
Vol. 42, No. 2, 1988
tended to inhibit the increased rate of the glucose output induced by epinephrine, compared to controls, but it was not significant (Fig. i). However, 1 m M DG5128 produced a significant decrease in the activated glucose output elicited by epinephrine. In addition, glucose output expressed as integrated increments from the rate of glucose output before the addition of DG5128 were -105 ± 30 and -339 ± 32 ~M/g.liver in the 0.I m M and 1 m M DG5128 administration, respectively. These values were significantly lower than controls (108 ± 65 ~M/g.liver; P < 0.05 vs 0.i m M DG5128, P < 0.01 vs 1 m M DG5128) as s h o w n F i g u r e 4. On the other hand, the addition of 1.4 × 10-10M glucagon produced an obvious increase of the glucose output. The basal rate of g l u c o s e o u t p u t and the v a l u e at 6 m i n u t e s a f t e r g l u c a g o n administration was 78 ± II and 172 ± 15 ~M/g.liver, respectively. The rate and changes of the glucose output after the addition of DG5128 (0.I mM, 1 mM) were not significantly different from controls as shown Figure 2 and 4.
Th e simultaneous administration of 10-6M epinephrine and 1.4 x lO-IUM glucagon increased glucose output from the basal rate of 64 ± I0 ~M/g.liver to the rate of 310 ± 40 ~M/g.liver at 6 minutes after the administration. And the addition of 0.I m M or 1 m M DG5128 produced a significant decrease of the increased glucose output, when induced by both epinephrine and glucagon, compared to controls (Fig. 3). Expressed as integrated changes of the glucose output after the addition of the agent, the values d u r i n g the a d m i n i s t r a t i o n of 0.i m M and 1 m M D G 5 1 2 8 w e r e 264 ± 99 and -114 ± 94 ~M/g.liver, respectively, significantly lower than the 625 ± 109 ~M/g.liver of controls (P < 0.05 vs 0.i m M DG5128, P < 0.01 vs 1 m M DG5128), as s h o w n F i g u r e 4.
Epinephrinel0-~M Glucogon 1.4x10~°M
o----o Control (N=5) : = DG 0.1mM(N=6) ~ - . 4 DG 1 r a M ( N = 6 ) DG
400
~s
~-~"-~"~
rain.
16
r:
E
\
o
300
200
lOO
1
3
5
9
Fig.
3
Effects of DG5128 (e • 0.I m M , = & - - - - - & i mM) on the rise of g ~ c o s e output by both 10-UM epinephrine and 1.4 x 10-±UM glucagon from the perfused rat liver. Values are means ± SE, with the numbers of animals given in parenthesis. *Different from Control, P < 0.01.
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~2-Blocker Effect on Glucose Output
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The second experiment: Insulin infusion ten~e~ to lower the increased glucose o-~put induced by 1.4 x 10-~VM glucagon, but it was not significantly different from the control values. In addition, the a d m i n i s t r a t i o n of DG5128 did not produce any significant changes in the increased glucose output by 1.4 x 10-~VM glucagon in the absence and presence of insulin. The administration of 7.0 x 10-11M glucagon produced al~Rst the same increase of the glucose output as that of 1.4 x 10-±UM glucagon, as shown Table I. In this study, however, the insulin infusion inhibited significantly t h ~ i n c r e a s e d rate of the glucose output induced by 7.0 x 10-~IM glucagon. Glucose output expressed as integrated increments from the rate of the glucose output before the addition of glucagon was 1683 ± 108 pM/g.liver in the presence of insulin, significantly lower than the value of 2064 ± Iii ~M/g.liver in control experiment. The addition of DG5128 in the absence of i n s ~ i n did not affect the glucose output elicited by 7.0 x 10-~IM glucagon, but decreased further the 7 . 0 x 10-±±M glucagon-induced increase of the glucose output when insulin was perfused (Table I).
~neph~
OOI
Glucogon
F~oine~rinepl~ C 4 ~
600 UM 4
2OO
T 0
~
[X;O.~rnMDGlrn~ Control OGIllr~ lY~ImM
-200
Con(rol
DG01rnM
Control~
Fig. 4 Changes in gluco#~ output induced by 10-6M epinephrine and/or 1.4 x 10 -I M glucagon after the administration of DG5128 in the perfused rat liver. Values are means ± SE. *,**Different from each control P < 0.05, P < 0.01
134
~2-Blocker Effect on Glucose Output
Table I.
Vol. 42, No. 2, 1988
Effects of DG5128 and/or insulin on glucagon-induced glucose output. Glucose output
(HM/g.liver)
Basal rate
Rate at 8 min
(N)
Net increase for I0 minutes
Glucagon 1.4 x 10-10M only + DG5128 + Insulin + DG5128 + Insulin
(5) (4) (4) (4)
87 85 73 93
± 7 ± 9 ± 12 ± i0
380 375 346 358
± ± ± ±
31 28 13 30
2174 2056 1839 1866
i 191 ± 125 ± 98 i 135
Glucagon ~ 7.0 x 10-±~M only + DG5128 + Insulin + DG5128 + Insulin
(5) (4) (4) (4)
62 72 70 72
± I0 ± I0 ± 19 ± 9
363 346 297 222
± ± ± ±
21 16 20* 36 *,+
2064 1823 1683 1080
± ± ± ±
iii 120 108" 133 **,+
Glucagon was added 5 min after the beginning of sampling as described in Materials and Methods. Insulin (i mU/ml) and D G 5 1 2 8 (I mM) w e r e a d d e d I0 m i n and 5 m i n b e f o r e the infusion of glucagon, respectively. Basal rate shows the level of glucose output before glucagon infusion and rate at 8 min indicates the level 8 min after glucagon infusion. Net increase in glucose output above the basal levels was c u m u l a t e d for a i0 m i n p e r i o d a f t e r the a d d i t i o n of glucagon. *P < 0.05, **P < 0.01 vs g l u c a g o n 7.0 x 1 0 - 1 1 M only +P < 0.05 vs glucagon + insulin Discussion Epinephrine and glucagon at physiological concentrations are well k n o w n to activate glucose production from the liver (11,12, 13). In the present study with the perfused rat liver, the increase in glucose production induced by these two hormones must be caused by glycogenolysis, since the liver perfusion was carried out in the absence of any gluconeogenic substrates. The increased rate of the glucose output is greater when both epinephrine and glucagon are administered together than when either hormone is administered alone. The present results are consistant with the work of Studer et al. using perfused hepatocytes (14). They found that the simultaneous administration of the two hormones caused an increase in phosphorylase activity which was quantitatively additive. In the present study, the administration of DG5128, an ~p-adrenergic antagonist, reduced the increased glucose output elicited by epinephrine alone or epinephrine plus glucagon. The effects of epinephrine on the carbohydrate m e t a b o l i s m in the liver are mediated by ~ and 6 adrenergic receptors. Rizza et al. (15) have reported that the stimulation of glucose production and the suppression of glucose clearance by epinephrine in man is predominantly the result of 6-
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~2-Blocker Effect on Glucose Output
135
adrenergic mechanisms. On the other hand, it has been investigated that the effects of epinephrine on the hepatic carbohydrate m e t a b o l i s m in rats are mediated predominantly by ~adrenergic receptors (10,16). The differences in species and experimental methods may account for the contradictory results. Recently, ~-adrenergic receptors have been subdivided into two types, ~i and ~p (17). Several investigators have suggested that the ~l-adrenerg~c action in the liver is mediated through an elevation of cytosolic Ca Z+ and that the ~2-adrenergic action is dependent on the inhibition of adenylate cyclase (18). However, Studer et al. (14) have reported that there is no evidence of an ~2-adrenergic inhibition of adenylate cyclase. These conflicting reports indicate that the mechanism of ~p-adrenergic action has not yet been clarified. Aggerback et alZ (19) have shown that prazocin and yohimbine, which are ~I- and ~p -adrenergic antagonists, respectively, produced an inhiSition of the glycogen phosphorylase stimulated by epinephrine in isolated rat hepatocytes. Therefore, it was suggested that the inhibition of the increased glucose output by DG5128 may be produced through a decrease of phosphorylase activity, as well as by yohimbine. On the o t h e r hand, it is well k n o w n that g l u c a g o n is important for the maintenance of the glucose level at the physiological condition (20), and this hormone plays an important role in the hyperglycemia of diabetes mellitus (21). In the present study, we have observed that glucose prp~uctions induced by @@ch of the two doses of glucagon (1.4 x 10-~UM and 7.0 x 1 0 - ~ M ) were almost the same. In addition, insulin infusion inhibits glucose production1~nduced by physiological concentration p~ glucagon (7.0 x 1 0 - ~ M ) , but not by higher dosage (1.4 x 10-±UM). These results have indicated that 7.0 x 10-±~M glucagon is sufficient for maximal glucose production by glucagon in this system and demonstrated further that insulin could not suppress the glucose production elicited by the higher concentration of glucagon (1.4 x 10-~UM). Kameda et al. have reported that DG5128 fails to decrease the elevated blood glucose in the streptozotocin-induced diabetic rats (2). The rats are regarded, however, as a model of insulindependent diabetes mellitus (IDDM), because the blood glucose levels in these rats are above 300-400 mg/dl, indicating that these rats are at the severe insulin deficient state. It is well known that the hypoglycemic agents such as sulfonylureas are ineffective for severe insulinopenic patients. Therefore, even if DG5128 does not decrease the blood glucose levels in these rats, we can not rule out the p o s s i b i l i t y of a h y p o g l y c e m i c effect of the agent. Furthermore, the present observation, in which DG5128 produces an inhibitory effect on the glucose output induced by physiological concentration of glucagon only in the presence of insulin, in addition to its stimulation of insulin secretion, leads to support the possibility that the substrate could be a hypoglycemic agent for the treatment of non-insulin dependent diabetes mellitus (NIDDM) like sulfonylureas. Acknowledgements We are grateful to Ms. H. Tachikawa preparation of this manuscript.
for aid in the
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