- Email: [email protected]
Gluconeogenesis in rabbit liver
Biochimica et Biophysics Acta, 675 (1981) 309-315 Elsevier/NorthHolland Biomedical Press
309
BBA 29639
GLUCONEOGENESIS IN RABBIT LIVER IV. THE EFFECTS OF GLUCAGON, EPINEPHRINE, cy-AND /3-ADRENERGICAGENTS ON GLUCONEOGENESIS AND PYRUVATE KINASE IN HEPATOCYTES GIVEN DIHYDROXYACETONE OR FRUCTOSE MARK A. YOREK a, GERALD A. RUFO, Jr. a**, JAMES B. BLAIR b and PAUL D. RAY a*** a Guy and Bertha Ireland Research Laboratory, Department of Biochemistry, Universityof North Dakota School of Medicine, Grand Forks, ND 58202 and b the Department of Biochemistry, West Virginia UniversityMedical Center, Morgantown, WV26506 (U.S.A.) (Received February 27th, 1981)
Key words: firuvate kinase; Gluconeogenesis; Dihydroxyacetone; Fructose; Adrenergic reagent; Glucagon; Epinephrine; (Rabbit liver)
1. Epinephtie, isoproterenol and phenylephrine each increases significantly gluconeogenesis (from dihydroxyacetone or D-fructose) and glycogenolysis when added to hepatocytes from 48-h fasted rabbits. Such stimulation of both processes by epinephrine, isoproterenol or phenylephrine is negated by the fl-adrenergicantagonist propranolol but remains significant in the presence of the ar-adrenergic antagonist phentolamine. Conversely, previous data suggest that catecholamine-induced stimulation of glucose formation from L-lactate is both 01-and @-adrenergic-sensitive. 2. Glucagon, epinephrine, isoproterenol, phenylephrine and dibutyryl cyclic AMP each inhibits significantly pyruvate kinase activity in rabbit hepatocytes. Inhibition of pyruvate kinase activity by epinephrine, isoproterenol or phenylephrine is negated by propranolol but insensitive to phentolamine. 3. These observations suggest that enhancement by epinephrine of glucose formation from either dihydroxyacetone or D-fructose is solely &adrenergic-regulated,just as is its inhibition of pyruvate kinase activity. Stimulation of gluconeogenesis by glucagon, epinephrine, isoproterenol, phenylephrine or dibutyryl cyclic AMP may be at least in part directly related to their ability to inhibit pyruvate kinase.
Introduction A number of recent studies have shown that the processes of gluconeogenesis and glycogenolysis are stimulated by either glucagon or catecholamines [l-7] and that stimulation of ‘gluconeogenesis is accompanied by a decrease in the activity of pyruvate kinase (EC 2.7.1.40) [8-121. Results from many
* Present address: Institute for Enzyme Research, Group II, University of Wisconsin, Madison, WI 5 3705, U.S.A. +* To whom correspondence should be addressed. 0 3044165/81/0000-0000/$02.50
@Elsevier/North-HoIIand
of these studies suggest that catecholamine-induced effects, while mediated by both (Y-and P-adrenergic stimulation, are primarily cw-adrenergic-mediated in rats [3,5,7,9-111; perhaps exclusively so in mature rats [ 12,131. Conversely, in vivo studies by Rizza et al. [14] suggest that catecholamine-induced glucose production in humans is predominantly fladrenergicrelated. Such divergent observations suggest a necessity for investigating the nature of catecholamineinduced alterations in carbohydrate metabolism in a variety of species, especially if a model of carbohydrate metabolism as it occurs in humans is being sought [15]. We recently published data which showed that Biomedical Press
310
gluconeogenesis in isolated rabbit hepatocytes given L-lactate while sensitive to both (Y-and /3-adrenergic stimulation is somewhat more /3-adrenergic-sensitive and that glycogenolysis in isolated perfused rabbit livers and hepatocytes from both young and mature rabbits is solely /3-adrenergic-related [ 16,171. Because of the sensitivity of lactate-derived gluconeogenesis as opposed to the insensitivity of’ glycogenolysis to a-adrenergic stimulation, it was of interest to investigate the effects of catecholamines on gluconeogenesis in rabbit hepatocytes given dihydroxyacetone or fructose. It was also of interest to see if stimulation by glucagon, catecholamines or dibutyryl cyclic AMP of gluconeogenesis in rabbit hepatocytes is accompanied by alteration in the activity of pyruvate kinase and if such alteration in the enzyme’s activity is, if observed in the case of catecholamines, a- and/or P-adrenergicmediated. These studies have been conducted and the results are contained herein.
Materials and Methods Animals. Male, New Zealand rabbits obtained from Klubertanz, Edgerton, WI, were maintained on tap water and Purina Rabbit Chow (unless fasted). Small 7-8-week-old rabbits weighing 1-I .2 kg were fasted for 48 h and used as liver donors; larger 2.5-3.0 kg rabbits were fasted for 24 h and used as blood donors. Techniques for liver perfusion and cell preparation. The techniques used for anesthetization of the liver and blood donors for liver perfusion and for isolation of hepatocytes have been described previously
Dbl.
Protocol for incubations. The experimental designs for incubations intended for measurement of glucose, glycogen and metabolites have been described previously [ 16,171. For incubations to be assayed for pyruvate kinase activity, 4.5 ml of Krebs-Henseleit HCOi buffer containing ‘fatty acid-poor’ bovine serum albumin and substrate were pre-incubated at 37°C for 5 min prior to addition of 4.5 ml of cells (about 5 * lo7 cells). Cells were incubated in the absence or presence of adrenergic antagonists for 5 mm at which time effecters were added and incubations carried out for 0 or I5 min at 37°C. The flasks’ contents were continuously oxygenated throughout the total period of incubation. The concentrations of
the various hormones, agonists and antagonists used were the same as used by Yorek et al. [ 161. Preparation of samples for assa,v of p.yruvate kinase. The flasks’ contents intended for assay of pyruvate kinase were, after 1.5 min of incubation, mixed with 18 ml of saturated (NH4)*S04 containing 30 mM 8-mercaptoethanol and centrifuged. After decantation of supernates, pellets were resuspended in 66% (NH4)2S0,, containing 30 mM P-mercaptoethanol. In some cases, liver samples were directly homogenized in 0.25 M sucrose containing 30 mM B-mercaptoethanol and centrifuged. Supernates were decanted and solid (NH4)?SOI, was added to the supernates to a final concentration of 66%. Assay for pyruvate kinase activity. Pyruvate kinase activity was assayed at 25°C by a calorimetric procedure essentially as described by Blair et al. [18]. Hepatocyte extract (25 ~1) was incubated for 15 min with 0.5 ml of 140 mM Tris-HCl (pH 7.5) containing 1% (w/v) Triton X-100, 66 mM KCl, 10 mM MgSO,, 2.5 mM ADP and 0.8 mM phosphoenolpyruvate in both the absence (-) and presence (+) of 200 PM fructose 1,6-bisphosphate. The reaction was terminated by addition of 0.5 ml 1% (w/v) dinitrophenylhydrazine in 2 M HCl. After sitting at room temperature for 10 min, color was developed by adding 1 .O ml 1.5 M NaOH and the absorbance at 520 nm was read 20 mm later. Blanks for enzyme incubated in the absence of phosphoenolpyrubate were obtained for each sample since some variations in this blank value were observed from sample to sample. The color development at 520 nm was linear with respect to pyruvate concentrations and time over the conditions applied in this investigation. The ratio (-/t) of pyruvate kinase activity measured in the absence (-)/presence (+) of fructose bisphosphate was determined. Absolute levels of pyruvate kinase activity for each liver preparation were also determined by direct assays coupled with lactate dehydrogenase as previously described [ 181. Analyses. Glycogen and hepatic metabolites were quantitated as described previously [ 161. D-Glucose was assayed by enzymatic conversion to 6-phosphogluconos-lactone [ 191. Expression of data and statistical analyses. Data obtained with hepatocytes are reported as means +I S.E. of rates of gluconeogenesis or glycogenolysis in pmol/30 min per 10s cells. Pyruvate kinase data
311 greater extent than from L-lactate, although all three effectors significantly enhance gluconeogenesis from all three substrates [16]. We also reported that gluconeogenesis from L-lactate is enhanced by both aand fl-adrenergic agonists and that the increase in glucose formation from lactate caused by catecholamines is both a- and fl-adrenergic-mediated. Because of these aforementioned observations and because dihydroxyacetone and D-fructose each enters into the pathway of gluconeogenesis at different sites and at sites considerably closer to glucose, we investigated the influences of ~- and /3-adrenerglc agonists and antagonists and also the adrenerglc nature of catecholamines' effects on gluconeogenesis and glycogenolysis in cells given dihydroxyacetone or D-fructose. Data presented in Table I show that addition of epinephrine, phenylephrine or isoproterenol to cells given dihydroxyacetone stimulates significantly the rates of gluconeogenesis and glycogenolysis compared to rates observed in the presence of substrate alone. Data also show that epinephrine's stimulation of glu-
are reported as means -+1 S.E. o f the activity ratio ( - / + ) measured in the absence ( - ) or presence (+) of 200 /aM fructose 1,6-bisphosphate and as times control. All P values were calculated from raw data and analyzed by Student's paired 't'-test. Materials. Sources of chemicals, substrates, enzymes, hormones, a- and/3-adrenergic agonists and antagonists have been reported previously [16]. Glucagon was a gift from Dr. William W. Bromer, Eli Lily; phentolamine-HC1 a gift from Dr. Richard H. Fair, Ciba; phenoxybenzamine-HC1 from Dr. John Atikinson, Smith, Kline and French.
Results
Influence of epinephrine, a- and ~-adrenergic agonists and antagonists on gluconeogenesis and glycogenolysis in isolated hepatocytes given 20 mM dihydroxyacetone We previously reported that glucagon, epinephrine and dibutyryl cyclic AMP each enhances gluconeogenesis from dihydroxyacetone and D-fructose to a
TABLE 1 INFLUENCES OF EPINEPHRINE, ~- AND fl-ADRENERGIC AGONISTS AND ANTAGONISTS ON GLUCONEOGENESIS AND GLYCOGENOLYSIS IN RABBIT HEPATOCYTES GIVEN20 mM DIHYDROXYACETONE Data were obtained from eight different preparations of hepatocytes from 48-h-fasted rabbits. Data for gluconeogenesis are expressed as means ± S.E. of izmol glucose/30 min per 108 cells and are based on the glucose appearing in the medium minus the amount of glucose contributed by glycogenolysis. Data for glycogenolysis are expressed as means ± S.E. of ~mol glucose/30 rain per 108 cells and are based on actual changes in glycogen content. All P values were calculated according to Student's paired t-test and data obtained with effector(s) tested vs data obtained without effector(s). Concentrations of additions were: epinephrine, 5 • 104 M; phenylephrine, 2 • 10-s M; isoproterenol, 104-6 M; propranolol, l0 -s M; phentolamine, 2 • 10 -s M Addition(s)
Glu¢oneogenesis
Glycogenolysis
Dihydroxyacetone +Epinephrine +Phenylephrine +Isoproterenol +Propranolol +Phentolamine
3.38 5.86 5.09 5.01 3.17 3.25
± 0.58 ± 0.74, P < 0.001 ± 0.88, P < 0.005 ± 0.69,P < 0.001 ± 0.63 ± 0.57
0.39 1.59 1.21 1.20 0.46 0.40
± 0.30 ± 0.48, P < 0.02 ± 0.34,P < 0.02 ± 0.36,P < 0.05 ± 0.32 ± 0.29
3.18 4.17 5.94 3.25 3.57 4.11 4.55 3.06
± 0.70 ± 0.79 ± 1.18, P < 0.01 ± 0.61 ± 0.56 ± 0.77 ± 0.88,P < 0.02 ± 0.63
0.38 0.74 2.16 0.63 0.54 0.60 1.06 0.60
± 0.31 ± 0.29 ± 0.78, P < 0.025 ± 0.29 ± 0.29 ± 0.30 ± 0.30,P < 0.005 ± 0.29
+Propranolol + phentolamine +Epinephrine + propranolol +Epinephrine + phentolamine +Epinephrine + propranolol + phentolamine +Isoproterenol + propranolol +Phenylephrine + ptopranolol +Phenylephrine + phentolamine +Phenylephrine 4- propranolol + phentolamine
312
coneogenesis and glycogenolysis above control rates remains significant in the presence of the a-adrenergic antagonist phentolamine but loses significance in the presence of the /3-adrenergic antagonist propranolol or a combination of both antagonists. Enhancement of gluconeogenesis and glycogenolysis by isoproterenol and phenylephrine also loses significance in the presence of propranolol whereas enhancement of these two processes by phenylephrine remains significant in the presence of phentolamine. We have previously shown that propranolol or phentolamine alone or in combination exert no significant effects on the stimulation by dibutyryl cyclic AMP of lactate’s conversion to glucose, indicating that these compounds do not exert non-specific deleterious effects on glucose formation [ 161.
D-fructose significantly increases rates of gluconeogenesis and glycogenolysis compared to control rates observed in the presence of D-fructose alone. Enhancement above control rates of gluconeogenesis and glycogenolysis by addition of epinephrine or phenylephrine remains significant in the presence of phentolamine but loses significance in the presence of propranolol or a combination of both antagonists. Enhancement of glucose formation by isoproterenol likewise loses significance in the presence of propranolol.
Influence of epinephrine, (Y-and Padrenerg’c agonists and antagonists on gluconeogenesis and glycogenolysis in isolated hepatocytes given 30 mM D-fructose Data presented in Table II show that epinephrine, phenylephrine or isoproterenol added to cells given
Influence of glucagon, epinephrine, dibutyryl cyclic AMP, a- and S-adrenergic agonists and antagonists on metabolite profiles of hepatocytes given dihydroxyacetone or D-fructose Other investigators have observed that addition of glucagon, epinephrine or cyclic AMP to isolated rat livers or hepatocytes given various substrates results in metabolite profiles characterized by higher than normal levels of phosphoenolpyruvate and less than normal levels of lactate and pyruvate [ 1,2,9,10,20-
TABLE II INFLUENCES OF EPINEPHRINE, cy- AND fl-ADRENERGIC AGONISTS AND ANTAGONISTS ON GLUCONEOGENESIS AND GLYCOGENOLYSIS IN RABBIT HEPATOCYTES GIVEN 30 mM DFRUCTOSE Data were obtained from eight different preparations of hepatocytes from 48-h-fasted rabbits. Data for gluconeogenesis are expressed as means f S.E. of pmol glucose/30 min per 10’ cells and are based on the quantity of glucose appearing in the medium minus the amount of glucose contributed by glycogenolysis. Data for glycogenolysis are expressed as means f S.E. of Mmol glucose/30 min. per lo* cells and are based on actual changes in glycogen content. All P values were calculated according to Student’s paired r-test and data obtained with effector tested vs data obtained without effector( Concentrations of additions were as in Table I Addition(s)
Gluconeogenesis
Glycogenolysis
D-Fructose +Epinephrine +Phenylephrine +Propranolol +Phentolamine +Propranolol + phentolamine +Epinephrine + propranolol +Epinephrine + phentolamine +Epinephrine + propranolol + phentolamine +Isoproterenol + propranolol +Phenylephrine + propranolol +Phenylephrine + propranolol +Phenylephrine + phentolamine +Phenylephrine + propranolol + phentolamine
5.24 9.45 7.28 10.50 5.50 5.13 4.69 5.42 10.10 4.88 5.50 5.56 6.85 4.65
0.56 1.18 0.89 1.61 0.57 0.59 0.58 0.73 1.63 0.66 0.57 0.61 0.97 0.67
+ 1.26 + 1.95, + 1.67, _+2.34, c 1.40 f 1.34 f 1.37 f 1.33 + 2.49, + 1.11 c 1.42 f 1.37 f 1.48, f 1.03
P < 0.001 P < 0.02 P < 0.005
P < 0.01
P < 0.005
+ 0.20 f 0.26, t 0.28, c 0.29, + 0.19 f 0.23 + 0.21 + 0.20 f 0.34, f 0.23 * 0.19 f 0.20 f 0.29, f 0.26
P < 0.005 P < 0.025 P < 0.001
P < 0.001
P < 0.02
313 221; in other words, a metabolic ‘crossover’, sug-
cagon, epinephrine or cyclic AMP inhibits pyruvate kinase activity from rat hepatocytes when assayed in the presence of subsaturating quantities of phosphoenolpyruvate and in the absence of fructose 1,6bisphosphate [SJO-12,181 and that the inhibition exerted by epinephrine is a-adrenergic-related. The activity ratios (--I+) of pyruvate kinase (assayed with 0.8 mM phosphoenolpyruvate in the absence (-) or presence (t) of 200 PM fructose 1,6-bisphosphate) in rabbit hepatocytes given dihy droxyacetone and incubated in the absence or presence of various effecters or combinations thereof are presented in Table III; data obtained in the presence of effecters are also expressed as % control. It is apparent that glucagon, dibutyryl cyclic AMP, epinephrine, isoproterenol and phenylephrine each significantly decreases total pyruvate kinase activity by 25-35%. It is also readily apparent that propranolo1 negates the inhibition of pyruvate kinase by epinephrine, isoproterenol or phenylephrine whereas phentolamine exerts essentially no influence on the inhibition caused by epinephrine or phenylephrine.
gestive of a decrease in the activity of pyruvate kinase. Although data are not presented we find that the metabolite profiles of rabbit hepatocytes given either dihydroxyacetone or D-fructose and subsequently given glucagon, epinephrine, dibutyryl cyclic AMP, isoproterenol or phenylephrlne when compared to control profiles of cells given substrate alone are consistently characterized by elevated levels of phosphoenolpyruvate and less than normal levels of pyruvate and lactate. We further find that the ‘crossover’ between phosphoenolpyruvate and pyruvate caused by the presence of epinephrine, isoproterenol or phenylephrine is consistently negated by propranolol but remains obvious in essentially all cases in the presence of phentolamine . Influence of g/wagon, epinephrine, dibutyryl cyclic AMP, OLand fl-adrenergic agonists and antagonists on the activity of pyruvate kinase
A number of investigators have reported that glu-
TABLEIII INFLUENCESOF GLUCAGON,DIBUTYRYLCYCLICAMP,EPINEPHRINE,OLAND@-ADRENERGIC AGONISTSANDANTAGONISTSONPYRUVATEKINASEACTIVITYIN RABBITHEPATOCYTES Data are expressed as means f S.E. of the ratio (-I+) of the activity of pyruvate kinase measured in the absence or presence of 200 PM fructose 1,6_bisphosphate. The number of different pairs of observations is shown in parentheses. Ail P values were cab culated according to Student’s paired r-test and data obtained with alI effecters or combinations thereof tested vs data obtained without effecters (that is in the presence of 20 mM dihydroxyacetone). Concentrations of additions were: ghrcagon, 5 . 10m8 M; dibutyryl cyclic AMP, 5 * lo4 M; epinephrine, 2.5 * 10m5 M; isoproterenol, 5 . 10” M; phenylephrine, lo4 M; propranolol, 5 . 10W5M; phentolamine, lo4 M Addition(s)
Activity ratio
Dihydroxyacetone (8) +Glucagon (8) +Dibutyryl cyclic AMP (7) +Epinephrine (8) +Isoproterenol(8) +Phenylephrine (7) +Propranolol(8) +PhentoIamine (8) +RopranoIol + phentolamine (7) +Epinephrine + propranolol(8) +Epinephrine + phentolamine (7) +Epinephrine + propranolol + phentolamine (7) +IsoproterenoI + propranolol(8) +PhenyIephrine + propranolol(7) +Phenylephrine + phentoIamine (7) +Phenylephrine + propranolol + phentohunine (7)
0.44 0.32 0.27 0.34 0.30 0.32 0.45 0.41 0.39 0.46 0.30 0.39 0.44 0.42 0.30 0.40
f 0.05 f 0.03,P f 0.04, P f 0.04, P f 0.05, P f 0.05, P f 0.05 f 0.05 f 0.05 f 0.04 f 0.03, P f 0.05 f 0.05 f 0.04 f 0.04, P f 0.05
% Control
< < < < <
0.01 0.01 0.01 0.01 0.01
< 0.01
< 0.01
100 69.2 65.0 77.1 70.3 78.6 101.4 91.8 94.3 104.4 66.3 92.4 97.9 103.3 71.0 96.1
f 6.3 f 3.6 f 4.4 * 6.1 * 4.1 f 2.4 f 2.3 f 2.8 f 2.4 f 3.9 f 4.0 f 1.5 f 2.8 f 3.3 f 1.9
314 Discussion
Many early in vivo observations indicated that glucagon and catecholamines stimulate glucose formation in a variety of species [23,24] and that such stimulation by catecholamines is ~-adrenergic-related and cyclic AMP-dependent [25-29]. More recent observations made with perfused rat livers and hepatocytes have established the enhancement by glucagon and catecholamines of glycogenolysis and ghiconeogenesis in this particular species [1-7] but have also led to the conclusion that enhancement of these processes by catecholamines is predominantly a-adrenergic-related and perhaps independent of cyclic AMP [3,5,7,9-13]. Since few studies have been performed with in vitro preparations from species other than rats, we have been performing such studies with perfused rabbit livers and hepatocytes. We reported previously that glucagon, epinephrine, norepinephrine, dibutyryl cyclic AMP, isoproterenol and phenylephrine each stimulates glycogenolysis and also gluconeogenesis in rabbit hepatocytes given 10 mM L-lactate [16,17]. We also reported that catecholamine-induced stimulation of glucose formation from lactate is both c~- and ~-adrenergic-sensitive whereas stimulation of glycogenolysis by catecholamines is solely ~3-adrenergic-sensitive [16,17]. Data presented in Tables I and II confirm our previous findings that gtucagon, epinephrine, dibutyryl cyclic AMP, isoproterenol and phenylephrine each stimulates glycogenolysis and also show that each of these effectors enhances gluconeogenesis in rabbit hepatocytes given 20 mM dihydroxyacetone or 30 mM D-fructose. Most interestingly, data in Tables I and II also indicate that epinephrine's stimulation of gluconeogenesis from these two substrates is essentially only j3-adrenergic-related. The observation that epinephrine's stimulation of ghiconeogenesis from lactate is both a- and ~-adrenergic-related whereas its enhancement of glucose formation from dihydroxyacetone or fructose is solely /3-adrenergic-sensitive suggests that an a-adrenergic-sensitive component exists between the sites of entry of L-lactate and dihydroxyacetone into the pathway of glucose formation although we have not identified such a component as yet. Many investigators have reported that enhancement by glucagon, catecholamines or cyclic AMP of
gluconeogenesis in rat livers or hepatocytes is accompanied by a decrease in the activity of hepatic pyruvate kinase which presumably decreases recycling of phosphoenolpyruvate back to pyruvate, thereby enhancing carbon flow toward glucose [8-12]. Such inhibition of pyruvate kinase by catecholamines appears to be predominantly a-adrenergic-mediated in rats, especially in mature rats, although controversy exists as to whether or not the a-adrenergic-mediated effects are cyclic AMP-dependent [30]. Data in Table III indicate that glucagon, catecholamines and dibutyryl cyclic AMP cause an approximate 30% inhibition of total pyruvate kinase activity in hepatocytes from fasted rabbits and that such inhibition by tors found to inhibit pyruvate kinase activity also cause a 'metabolic crossover', as indicated by elevated concentrations of phosphoenolpyruvate and less than normal concentrations of pyruvate and lactate, thus lending support to the idea that enhancement of gluconeogenesis in rabbit liver by glucagon and catecholamines is at least in part due to a cyclic AMP-dependent inhibition of pyruvate kinase activity. Interestingly, we find that about 70% of the total pyruvate kinase activity in rabbit liver homogenates or hepatocytes is neutralized by goat anti-rat type L pyruvate kinase serum, suggesting the presence of a considerable amount of another type or types of pyruvate kinase activity. We found that 74-+ 1.1% and 67 +- 1.8% of the total pyruvate kinase activity in rabbit liver homogenates and hepatocytes, respectively, is neutralized by goat anti-rat type L pyruvate kinase serum. Our anti-type L serum inhibits Sigma rabbit type L enzyme by 95% and inhibits our preparation of rat type L enzyme by more than 95%. Conversely, our anti-serum inhibits Sigma rabbit type M1 pyruvate kinase by only 1.3%. Our data thus quaIitatively agree with the findings of JOhnson and Veneziale [31] although they report that only 4 0 60% of pyruvate kinase activity in rabbit liver is inhibited by anti-type L 3,-globulin and all but 5% of the remaining activity is inhibited by anti-type M "),-globulin. We have not directly ascertained as yet whether or not effector-mediated inhibition of pyruvate kinase such as reported in Table III is restricted solely to the type L enzyme in rabbit liver as it is in rats [32]. However, if only the type L enzyme of rabbit hepatocytes is inhibited by glucagon, catecholamines and dibutyryl cyclic AMP, then inhibition of
315 the type L enzyme is about 4 5 - 5 0 % (as opposed to 30% inhibition of total pyruvate kinase activity reported in Table III) which more closely approaches the amount of inhibition ( 5 0 - 8 0 % ) by these effectors of total pyruvate kinase activity (which is essentially only type L enzyme) in rat hepatocytes [8,9, 11,33,34]. Acknowledgements These studies were supported by grants to P.D.R. from the National Institutes of Health (AM 12705) and the North Dakota Affiliate of the American Diabetes Association. J.B.B. is a recipient of a Research Career Development Award (AM 00428) and his studies were supported by a grant from the National Institutes of Health (AM 28176). A part of the data in this paper was taken from a dissertation submitted by M.A.Y. to the Graduate School of the University of North Dakota. References 1 Veneziale, C.M. (1971) Biochemistry 10, 3443-3447 2 Veneziale, C.M. (1972) Biochemistry 11, 3286-3289 3 Tolbert, M.E.M., Butcher, F.R. and Fain, J.N. (1973) J. Biol. Chem. 248, 5686-5692 4 Garrison, J.C. and Haynes, R.C., Jr. (1973) J. Biol. Chem. 248,5333-5343 5 Tolbert, M.E.M. and Fain, J.N. (1974) J. Biol. Chem. 249, 4462-4466 6 Exton, J.H. and Harper, S.C. (1975) Adv. Cyclic Nucleotide Res. 5,519-532 7 Fain, J.N., Tolbert, M.E.M., Pointer, R.H., Butcher, F.R. and Arnold, A. (1975) Metabolism 24,395-407 8 Feliu, J.E., Hue, L. and Hers, H.B. (1976) Proe. Natl. Acad. Sci. U.S.A. 73, 2762-2766 9 Pilkis, S.J., Riou, J.P. and Claus, T.H. (1976) J. Biol. Chem. 251, 7841-7852 10 Chan, T.M. and Exton, J.H. (1978) J. Biol. Chem. 253, 6393-6400
11 Foster, J.L. and Blair, J.B. (1978) Arch. Biochem. Biophys. 189,263-276 12 Blair, J.B., James, M.E. and Foster, J.L. (1979) J. Biol. Chem. 254, 7585-7590 13 Blair, J.B., James, M.E. and Foster, J.L. (1979) J. Biol. Chem. 254, 7579-7584 14 Rizza, R.A., Cryer, P.E., Haymond, M.W. and Gerich, J.E. (1980) J. Clin. Invest. 65,682-689 15 Hanson, R.W. (1980) Trends Biochem. Sci. 5, l - I I 16 Yorek, M.A., Rufo, G.A., Jr. and Ray, P.D. (1980) Bioclaim. Biophys. Acta 632, 517-526 17 Rufo, G.A., Jr., Yorek, M.A. and Ray, P.D. (1981) Biochim. Biophys. Acta 674,307-315 18 Blair, J.B., Cimbala, M.A., Foster, J.L. and Morgan, R.A. (1976) J. Biol. Chem. 251, 3756-3762 19 Bergmeyer, H.U. (1965) Methods of Enzymatic Analysis, 1st edn., pp. 117-123, Academic Press, New York 20 Exton, J.H. and Park, C.R. (1969) J. Biol. Chem. 244, 1424-1433 21 Blair, J.B., Cook, D.E. and Lardy, H.A. (1973) J. Biol. Chem. 248, 3601-3607 22 Clark, M.G., Kneel N.M., Bosch, A.L. and Lardy, H.A. (1974) J. Biol. Chem. 249, 5695-5703 23 Sutherland, E.W. (1950) Recent Prog. Horm. Res. 5, 441-463 24 Ellis, S. (1956) Pharmacol. Rev. 8,485-562 25 Sutherland, E.W. and RaU, T.W. (1960) Pharmacol. Rev. 12,265-299 26 Sutherland, E.W. and Robison, G.A. (1969) Diabetes 18, 797-819 27 Exton, J.H., Mallette, L.E., Jefferson, L.S., Wong, E.H.A., Friedmann, N. and Park, C.R. (1970) Am. J. Clin. Nutr. 23,993-1003 28 Robison, G.A., Butcher, R.W. and Sutheriand, E.W. (1967) Ann. N.Y. Acad. Sci. 139,703-723 29 Hornbrook, K.R. (1970) Fed. Proc. 29, 1381-1385 30 Kemp, B.E. and Clark, M.G. (1978) J. Biol. Chem. 253, 5147-5154 31 Johnson, M.L. and Veneziale, C.M. (1980) Biochemistry 19, 2191-2195 32 Van Berkel, J.C., Koster, J.F. and Hiilsmann, W.C. (1972) Biochim. Biophys. Acta 276,425-429 33 Van Berkel, J.C., Kruizt, J.K. and Koster, J.F. (1977) Biochim. Biophys. Acta 500,267-276 34 Ishibashi, H. and Cottam, G.L. (1978) J. Biol. Chem. 253, 8767-8771
309
BBA 29639
GLUCONEOGENESIS IN RABBIT LIVER IV. THE EFFECTS OF GLUCAGON, EPINEPHRINE, cy-AND /3-ADRENERGICAGENTS ON GLUCONEOGENESIS AND PYRUVATE KINASE IN HEPATOCYTES GIVEN DIHYDROXYACETONE OR FRUCTOSE MARK A. YOREK a, GERALD A. RUFO, Jr. a**, JAMES B. BLAIR b and PAUL D. RAY a*** a Guy and Bertha Ireland Research Laboratory, Department of Biochemistry, Universityof North Dakota School of Medicine, Grand Forks, ND 58202 and b the Department of Biochemistry, West Virginia UniversityMedical Center, Morgantown, WV26506 (U.S.A.) (Received February 27th, 1981)
Key words: firuvate kinase; Gluconeogenesis; Dihydroxyacetone; Fructose; Adrenergic reagent; Glucagon; Epinephrine; (Rabbit liver)
1. Epinephtie, isoproterenol and phenylephrine each increases significantly gluconeogenesis (from dihydroxyacetone or D-fructose) and glycogenolysis when added to hepatocytes from 48-h fasted rabbits. Such stimulation of both processes by epinephrine, isoproterenol or phenylephrine is negated by the fl-adrenergicantagonist propranolol but remains significant in the presence of the ar-adrenergic antagonist phentolamine. Conversely, previous data suggest that catecholamine-induced stimulation of glucose formation from L-lactate is both 01-and @-adrenergic-sensitive. 2. Glucagon, epinephrine, isoproterenol, phenylephrine and dibutyryl cyclic AMP each inhibits significantly pyruvate kinase activity in rabbit hepatocytes. Inhibition of pyruvate kinase activity by epinephrine, isoproterenol or phenylephrine is negated by propranolol but insensitive to phentolamine. 3. These observations suggest that enhancement by epinephrine of glucose formation from either dihydroxyacetone or D-fructose is solely &adrenergic-regulated,just as is its inhibition of pyruvate kinase activity. Stimulation of gluconeogenesis by glucagon, epinephrine, isoproterenol, phenylephrine or dibutyryl cyclic AMP may be at least in part directly related to their ability to inhibit pyruvate kinase.
Introduction A number of recent studies have shown that the processes of gluconeogenesis and glycogenolysis are stimulated by either glucagon or catecholamines [l-7] and that stimulation of ‘gluconeogenesis is accompanied by a decrease in the activity of pyruvate kinase (EC 2.7.1.40) [8-121. Results from many
* Present address: Institute for Enzyme Research, Group II, University of Wisconsin, Madison, WI 5 3705, U.S.A. +* To whom correspondence should be addressed. 0 3044165/81/0000-0000/$02.50
@Elsevier/North-HoIIand
of these studies suggest that catecholamine-induced effects, while mediated by both (Y-and P-adrenergic stimulation, are primarily cw-adrenergic-mediated in rats [3,5,7,9-111; perhaps exclusively so in mature rats [ 12,131. Conversely, in vivo studies by Rizza et al. [14] suggest that catecholamine-induced glucose production in humans is predominantly fladrenergicrelated. Such divergent observations suggest a necessity for investigating the nature of catecholamineinduced alterations in carbohydrate metabolism in a variety of species, especially if a model of carbohydrate metabolism as it occurs in humans is being sought [15]. We recently published data which showed that Biomedical Press
310
gluconeogenesis in isolated rabbit hepatocytes given L-lactate while sensitive to both (Y-and /3-adrenergic stimulation is somewhat more /3-adrenergic-sensitive and that glycogenolysis in isolated perfused rabbit livers and hepatocytes from both young and mature rabbits is solely /3-adrenergic-related [ 16,171. Because of the sensitivity of lactate-derived gluconeogenesis as opposed to the insensitivity of’ glycogenolysis to a-adrenergic stimulation, it was of interest to investigate the effects of catecholamines on gluconeogenesis in rabbit hepatocytes given dihydroxyacetone or fructose. It was also of interest to see if stimulation by glucagon, catecholamines or dibutyryl cyclic AMP of gluconeogenesis in rabbit hepatocytes is accompanied by alteration in the activity of pyruvate kinase and if such alteration in the enzyme’s activity is, if observed in the case of catecholamines, a- and/or P-adrenergicmediated. These studies have been conducted and the results are contained herein.
Materials and Methods Animals. Male, New Zealand rabbits obtained from Klubertanz, Edgerton, WI, were maintained on tap water and Purina Rabbit Chow (unless fasted). Small 7-8-week-old rabbits weighing 1-I .2 kg were fasted for 48 h and used as liver donors; larger 2.5-3.0 kg rabbits were fasted for 24 h and used as blood donors. Techniques for liver perfusion and cell preparation. The techniques used for anesthetization of the liver and blood donors for liver perfusion and for isolation of hepatocytes have been described previously
Dbl.
Protocol for incubations. The experimental designs for incubations intended for measurement of glucose, glycogen and metabolites have been described previously [ 16,171. For incubations to be assayed for pyruvate kinase activity, 4.5 ml of Krebs-Henseleit HCOi buffer containing ‘fatty acid-poor’ bovine serum albumin and substrate were pre-incubated at 37°C for 5 min prior to addition of 4.5 ml of cells (about 5 * lo7 cells). Cells were incubated in the absence or presence of adrenergic antagonists for 5 mm at which time effecters were added and incubations carried out for 0 or I5 min at 37°C. The flasks’ contents were continuously oxygenated throughout the total period of incubation. The concentrations of
the various hormones, agonists and antagonists used were the same as used by Yorek et al. [ 161. Preparation of samples for assa,v of p.yruvate kinase. The flasks’ contents intended for assay of pyruvate kinase were, after 1.5 min of incubation, mixed with 18 ml of saturated (NH4)*S04 containing 30 mM 8-mercaptoethanol and centrifuged. After decantation of supernates, pellets were resuspended in 66% (NH4)2S0,, containing 30 mM P-mercaptoethanol. In some cases, liver samples were directly homogenized in 0.25 M sucrose containing 30 mM B-mercaptoethanol and centrifuged. Supernates were decanted and solid (NH4)?SOI, was added to the supernates to a final concentration of 66%. Assay for pyruvate kinase activity. Pyruvate kinase activity was assayed at 25°C by a calorimetric procedure essentially as described by Blair et al. [18]. Hepatocyte extract (25 ~1) was incubated for 15 min with 0.5 ml of 140 mM Tris-HCl (pH 7.5) containing 1% (w/v) Triton X-100, 66 mM KCl, 10 mM MgSO,, 2.5 mM ADP and 0.8 mM phosphoenolpyruvate in both the absence (-) and presence (+) of 200 PM fructose 1,6-bisphosphate. The reaction was terminated by addition of 0.5 ml 1% (w/v) dinitrophenylhydrazine in 2 M HCl. After sitting at room temperature for 10 min, color was developed by adding 1 .O ml 1.5 M NaOH and the absorbance at 520 nm was read 20 mm later. Blanks for enzyme incubated in the absence of phosphoenolpyrubate were obtained for each sample since some variations in this blank value were observed from sample to sample. The color development at 520 nm was linear with respect to pyruvate concentrations and time over the conditions applied in this investigation. The ratio (-/t) of pyruvate kinase activity measured in the absence (-)/presence (+) of fructose bisphosphate was determined. Absolute levels of pyruvate kinase activity for each liver preparation were also determined by direct assays coupled with lactate dehydrogenase as previously described [ 181. Analyses. Glycogen and hepatic metabolites were quantitated as described previously [ 161. D-Glucose was assayed by enzymatic conversion to 6-phosphogluconos-lactone [ 191. Expression of data and statistical analyses. Data obtained with hepatocytes are reported as means +I S.E. of rates of gluconeogenesis or glycogenolysis in pmol/30 min per 10s cells. Pyruvate kinase data
311 greater extent than from L-lactate, although all three effectors significantly enhance gluconeogenesis from all three substrates [16]. We also reported that gluconeogenesis from L-lactate is enhanced by both aand fl-adrenergic agonists and that the increase in glucose formation from lactate caused by catecholamines is both a- and fl-adrenergic-mediated. Because of these aforementioned observations and because dihydroxyacetone and D-fructose each enters into the pathway of gluconeogenesis at different sites and at sites considerably closer to glucose, we investigated the influences of ~- and /3-adrenerglc agonists and antagonists and also the adrenerglc nature of catecholamines' effects on gluconeogenesis and glycogenolysis in cells given dihydroxyacetone or D-fructose. Data presented in Table I show that addition of epinephrine, phenylephrine or isoproterenol to cells given dihydroxyacetone stimulates significantly the rates of gluconeogenesis and glycogenolysis compared to rates observed in the presence of substrate alone. Data also show that epinephrine's stimulation of glu-
are reported as means -+1 S.E. o f the activity ratio ( - / + ) measured in the absence ( - ) or presence (+) of 200 /aM fructose 1,6-bisphosphate and as times control. All P values were calculated from raw data and analyzed by Student's paired 't'-test. Materials. Sources of chemicals, substrates, enzymes, hormones, a- and/3-adrenergic agonists and antagonists have been reported previously [16]. Glucagon was a gift from Dr. William W. Bromer, Eli Lily; phentolamine-HC1 a gift from Dr. Richard H. Fair, Ciba; phenoxybenzamine-HC1 from Dr. John Atikinson, Smith, Kline and French.
Results
Influence of epinephrine, a- and ~-adrenergic agonists and antagonists on gluconeogenesis and glycogenolysis in isolated hepatocytes given 20 mM dihydroxyacetone We previously reported that glucagon, epinephrine and dibutyryl cyclic AMP each enhances gluconeogenesis from dihydroxyacetone and D-fructose to a
TABLE 1 INFLUENCES OF EPINEPHRINE, ~- AND fl-ADRENERGIC AGONISTS AND ANTAGONISTS ON GLUCONEOGENESIS AND GLYCOGENOLYSIS IN RABBIT HEPATOCYTES GIVEN20 mM DIHYDROXYACETONE Data were obtained from eight different preparations of hepatocytes from 48-h-fasted rabbits. Data for gluconeogenesis are expressed as means ± S.E. of izmol glucose/30 min per 108 cells and are based on the glucose appearing in the medium minus the amount of glucose contributed by glycogenolysis. Data for glycogenolysis are expressed as means ± S.E. of ~mol glucose/30 rain per 108 cells and are based on actual changes in glycogen content. All P values were calculated according to Student's paired t-test and data obtained with effector(s) tested vs data obtained without effector(s). Concentrations of additions were: epinephrine, 5 • 104 M; phenylephrine, 2 • 10-s M; isoproterenol, 104-6 M; propranolol, l0 -s M; phentolamine, 2 • 10 -s M Addition(s)
Glu¢oneogenesis
Glycogenolysis
Dihydroxyacetone +Epinephrine +Phenylephrine +Isoproterenol +Propranolol +Phentolamine
3.38 5.86 5.09 5.01 3.17 3.25
± 0.58 ± 0.74, P < 0.001 ± 0.88, P < 0.005 ± 0.69,P < 0.001 ± 0.63 ± 0.57
0.39 1.59 1.21 1.20 0.46 0.40
± 0.30 ± 0.48, P < 0.02 ± 0.34,P < 0.02 ± 0.36,P < 0.05 ± 0.32 ± 0.29
3.18 4.17 5.94 3.25 3.57 4.11 4.55 3.06
± 0.70 ± 0.79 ± 1.18, P < 0.01 ± 0.61 ± 0.56 ± 0.77 ± 0.88,P < 0.02 ± 0.63
0.38 0.74 2.16 0.63 0.54 0.60 1.06 0.60
± 0.31 ± 0.29 ± 0.78, P < 0.025 ± 0.29 ± 0.29 ± 0.30 ± 0.30,P < 0.005 ± 0.29
+Propranolol + phentolamine +Epinephrine + propranolol +Epinephrine + phentolamine +Epinephrine + propranolol + phentolamine +Isoproterenol + propranolol +Phenylephrine + ptopranolol +Phenylephrine + phentolamine +Phenylephrine 4- propranolol + phentolamine
312
coneogenesis and glycogenolysis above control rates remains significant in the presence of the a-adrenergic antagonist phentolamine but loses significance in the presence of the /3-adrenergic antagonist propranolol or a combination of both antagonists. Enhancement of gluconeogenesis and glycogenolysis by isoproterenol and phenylephrine also loses significance in the presence of propranolol whereas enhancement of these two processes by phenylephrine remains significant in the presence of phentolamine. We have previously shown that propranolol or phentolamine alone or in combination exert no significant effects on the stimulation by dibutyryl cyclic AMP of lactate’s conversion to glucose, indicating that these compounds do not exert non-specific deleterious effects on glucose formation [ 161.
D-fructose significantly increases rates of gluconeogenesis and glycogenolysis compared to control rates observed in the presence of D-fructose alone. Enhancement above control rates of gluconeogenesis and glycogenolysis by addition of epinephrine or phenylephrine remains significant in the presence of phentolamine but loses significance in the presence of propranolol or a combination of both antagonists. Enhancement of glucose formation by isoproterenol likewise loses significance in the presence of propranolol.
Influence of epinephrine, (Y-and Padrenerg’c agonists and antagonists on gluconeogenesis and glycogenolysis in isolated hepatocytes given 30 mM D-fructose Data presented in Table II show that epinephrine, phenylephrine or isoproterenol added to cells given
Influence of glucagon, epinephrine, dibutyryl cyclic AMP, a- and S-adrenergic agonists and antagonists on metabolite profiles of hepatocytes given dihydroxyacetone or D-fructose Other investigators have observed that addition of glucagon, epinephrine or cyclic AMP to isolated rat livers or hepatocytes given various substrates results in metabolite profiles characterized by higher than normal levels of phosphoenolpyruvate and less than normal levels of lactate and pyruvate [ 1,2,9,10,20-
TABLE II INFLUENCES OF EPINEPHRINE, cy- AND fl-ADRENERGIC AGONISTS AND ANTAGONISTS ON GLUCONEOGENESIS AND GLYCOGENOLYSIS IN RABBIT HEPATOCYTES GIVEN 30 mM DFRUCTOSE Data were obtained from eight different preparations of hepatocytes from 48-h-fasted rabbits. Data for gluconeogenesis are expressed as means f S.E. of pmol glucose/30 min per 10’ cells and are based on the quantity of glucose appearing in the medium minus the amount of glucose contributed by glycogenolysis. Data for glycogenolysis are expressed as means f S.E. of Mmol glucose/30 min. per lo* cells and are based on actual changes in glycogen content. All P values were calculated according to Student’s paired r-test and data obtained with effector tested vs data obtained without effector( Concentrations of additions were as in Table I Addition(s)
Gluconeogenesis
Glycogenolysis
D-Fructose +Epinephrine +Phenylephrine +Propranolol +Phentolamine +Propranolol + phentolamine +Epinephrine + propranolol +Epinephrine + phentolamine +Epinephrine + propranolol + phentolamine +Isoproterenol + propranolol +Phenylephrine + propranolol +Phenylephrine + propranolol +Phenylephrine + phentolamine +Phenylephrine + propranolol + phentolamine
5.24 9.45 7.28 10.50 5.50 5.13 4.69 5.42 10.10 4.88 5.50 5.56 6.85 4.65
0.56 1.18 0.89 1.61 0.57 0.59 0.58 0.73 1.63 0.66 0.57 0.61 0.97 0.67
+ 1.26 + 1.95, + 1.67, _+2.34, c 1.40 f 1.34 f 1.37 f 1.33 + 2.49, + 1.11 c 1.42 f 1.37 f 1.48, f 1.03
P < 0.001 P < 0.02 P < 0.005
P < 0.01
P < 0.005
+ 0.20 f 0.26, t 0.28, c 0.29, + 0.19 f 0.23 + 0.21 + 0.20 f 0.34, f 0.23 * 0.19 f 0.20 f 0.29, f 0.26
P < 0.005 P < 0.025 P < 0.001
P < 0.001
P < 0.02
313 221; in other words, a metabolic ‘crossover’, sug-
cagon, epinephrine or cyclic AMP inhibits pyruvate kinase activity from rat hepatocytes when assayed in the presence of subsaturating quantities of phosphoenolpyruvate and in the absence of fructose 1,6bisphosphate [SJO-12,181 and that the inhibition exerted by epinephrine is a-adrenergic-related. The activity ratios (--I+) of pyruvate kinase (assayed with 0.8 mM phosphoenolpyruvate in the absence (-) or presence (t) of 200 PM fructose 1,6-bisphosphate) in rabbit hepatocytes given dihy droxyacetone and incubated in the absence or presence of various effecters or combinations thereof are presented in Table III; data obtained in the presence of effecters are also expressed as % control. It is apparent that glucagon, dibutyryl cyclic AMP, epinephrine, isoproterenol and phenylephrine each significantly decreases total pyruvate kinase activity by 25-35%. It is also readily apparent that propranolo1 negates the inhibition of pyruvate kinase by epinephrine, isoproterenol or phenylephrine whereas phentolamine exerts essentially no influence on the inhibition caused by epinephrine or phenylephrine.
gestive of a decrease in the activity of pyruvate kinase. Although data are not presented we find that the metabolite profiles of rabbit hepatocytes given either dihydroxyacetone or D-fructose and subsequently given glucagon, epinephrine, dibutyryl cyclic AMP, isoproterenol or phenylephrlne when compared to control profiles of cells given substrate alone are consistently characterized by elevated levels of phosphoenolpyruvate and less than normal levels of pyruvate and lactate. We further find that the ‘crossover’ between phosphoenolpyruvate and pyruvate caused by the presence of epinephrine, isoproterenol or phenylephrine is consistently negated by propranolol but remains obvious in essentially all cases in the presence of phentolamine . Influence of g/wagon, epinephrine, dibutyryl cyclic AMP, OLand fl-adrenergic agonists and antagonists on the activity of pyruvate kinase
A number of investigators have reported that glu-
TABLEIII INFLUENCESOF GLUCAGON,DIBUTYRYLCYCLICAMP,EPINEPHRINE,OLAND@-ADRENERGIC AGONISTSANDANTAGONISTSONPYRUVATEKINASEACTIVITYIN RABBITHEPATOCYTES Data are expressed as means f S.E. of the ratio (-I+) of the activity of pyruvate kinase measured in the absence or presence of 200 PM fructose 1,6_bisphosphate. The number of different pairs of observations is shown in parentheses. Ail P values were cab culated according to Student’s paired r-test and data obtained with alI effecters or combinations thereof tested vs data obtained without effecters (that is in the presence of 20 mM dihydroxyacetone). Concentrations of additions were: ghrcagon, 5 . 10m8 M; dibutyryl cyclic AMP, 5 * lo4 M; epinephrine, 2.5 * 10m5 M; isoproterenol, 5 . 10” M; phenylephrine, lo4 M; propranolol, 5 . 10W5M; phentolamine, lo4 M Addition(s)
Activity ratio
Dihydroxyacetone (8) +Glucagon (8) +Dibutyryl cyclic AMP (7) +Epinephrine (8) +Isoproterenol(8) +Phenylephrine (7) +Propranolol(8) +PhentoIamine (8) +RopranoIol + phentolamine (7) +Epinephrine + propranolol(8) +Epinephrine + phentolamine (7) +Epinephrine + propranolol + phentolamine (7) +IsoproterenoI + propranolol(8) +PhenyIephrine + propranolol(7) +Phenylephrine + phentoIamine (7) +Phenylephrine + propranolol + phentohunine (7)
0.44 0.32 0.27 0.34 0.30 0.32 0.45 0.41 0.39 0.46 0.30 0.39 0.44 0.42 0.30 0.40
f 0.05 f 0.03,P f 0.04, P f 0.04, P f 0.05, P f 0.05, P f 0.05 f 0.05 f 0.05 f 0.04 f 0.03, P f 0.05 f 0.05 f 0.04 f 0.04, P f 0.05
% Control
< < < < <
0.01 0.01 0.01 0.01 0.01
< 0.01
< 0.01
100 69.2 65.0 77.1 70.3 78.6 101.4 91.8 94.3 104.4 66.3 92.4 97.9 103.3 71.0 96.1
f 6.3 f 3.6 f 4.4 * 6.1 * 4.1 f 2.4 f 2.3 f 2.8 f 2.4 f 3.9 f 4.0 f 1.5 f 2.8 f 3.3 f 1.9
314 Discussion
Many early in vivo observations indicated that glucagon and catecholamines stimulate glucose formation in a variety of species [23,24] and that such stimulation by catecholamines is ~-adrenergic-related and cyclic AMP-dependent [25-29]. More recent observations made with perfused rat livers and hepatocytes have established the enhancement by glucagon and catecholamines of glycogenolysis and ghiconeogenesis in this particular species [1-7] but have also led to the conclusion that enhancement of these processes by catecholamines is predominantly a-adrenergic-related and perhaps independent of cyclic AMP [3,5,7,9-13]. Since few studies have been performed with in vitro preparations from species other than rats, we have been performing such studies with perfused rabbit livers and hepatocytes. We reported previously that glucagon, epinephrine, norepinephrine, dibutyryl cyclic AMP, isoproterenol and phenylephrine each stimulates glycogenolysis and also gluconeogenesis in rabbit hepatocytes given 10 mM L-lactate [16,17]. We also reported that catecholamine-induced stimulation of glucose formation from lactate is both c~- and ~-adrenergic-sensitive whereas stimulation of glycogenolysis by catecholamines is solely ~3-adrenergic-sensitive [16,17]. Data presented in Tables I and II confirm our previous findings that gtucagon, epinephrine, dibutyryl cyclic AMP, isoproterenol and phenylephrine each stimulates glycogenolysis and also show that each of these effectors enhances gluconeogenesis in rabbit hepatocytes given 20 mM dihydroxyacetone or 30 mM D-fructose. Most interestingly, data in Tables I and II also indicate that epinephrine's stimulation of gluconeogenesis from these two substrates is essentially only j3-adrenergic-related. The observation that epinephrine's stimulation of ghiconeogenesis from lactate is both a- and ~-adrenergic-related whereas its enhancement of glucose formation from dihydroxyacetone or fructose is solely /3-adrenergic-sensitive suggests that an a-adrenergic-sensitive component exists between the sites of entry of L-lactate and dihydroxyacetone into the pathway of glucose formation although we have not identified such a component as yet. Many investigators have reported that enhancement by glucagon, catecholamines or cyclic AMP of
gluconeogenesis in rat livers or hepatocytes is accompanied by a decrease in the activity of hepatic pyruvate kinase which presumably decreases recycling of phosphoenolpyruvate back to pyruvate, thereby enhancing carbon flow toward glucose [8-12]. Such inhibition of pyruvate kinase by catecholamines appears to be predominantly a-adrenergic-mediated in rats, especially in mature rats, although controversy exists as to whether or not the a-adrenergic-mediated effects are cyclic AMP-dependent [30]. Data in Table III indicate that glucagon, catecholamines and dibutyryl cyclic AMP cause an approximate 30% inhibition of total pyruvate kinase activity in hepatocytes from fasted rabbits and that such inhibition by tors found to inhibit pyruvate kinase activity also cause a 'metabolic crossover', as indicated by elevated concentrations of phosphoenolpyruvate and less than normal concentrations of pyruvate and lactate, thus lending support to the idea that enhancement of gluconeogenesis in rabbit liver by glucagon and catecholamines is at least in part due to a cyclic AMP-dependent inhibition of pyruvate kinase activity. Interestingly, we find that about 70% of the total pyruvate kinase activity in rabbit liver homogenates or hepatocytes is neutralized by goat anti-rat type L pyruvate kinase serum, suggesting the presence of a considerable amount of another type or types of pyruvate kinase activity. We found that 74-+ 1.1% and 67 +- 1.8% of the total pyruvate kinase activity in rabbit liver homogenates and hepatocytes, respectively, is neutralized by goat anti-rat type L pyruvate kinase serum. Our anti-type L serum inhibits Sigma rabbit type L enzyme by 95% and inhibits our preparation of rat type L enzyme by more than 95%. Conversely, our anti-serum inhibits Sigma rabbit type M1 pyruvate kinase by only 1.3%. Our data thus quaIitatively agree with the findings of JOhnson and Veneziale [31] although they report that only 4 0 60% of pyruvate kinase activity in rabbit liver is inhibited by anti-type L 3,-globulin and all but 5% of the remaining activity is inhibited by anti-type M "),-globulin. We have not directly ascertained as yet whether or not effector-mediated inhibition of pyruvate kinase such as reported in Table III is restricted solely to the type L enzyme in rabbit liver as it is in rats [32]. However, if only the type L enzyme of rabbit hepatocytes is inhibited by glucagon, catecholamines and dibutyryl cyclic AMP, then inhibition of
315 the type L enzyme is about 4 5 - 5 0 % (as opposed to 30% inhibition of total pyruvate kinase activity reported in Table III) which more closely approaches the amount of inhibition ( 5 0 - 8 0 % ) by these effectors of total pyruvate kinase activity (which is essentially only type L enzyme) in rat hepatocytes [8,9, 11,33,34]. Acknowledgements These studies were supported by grants to P.D.R. from the National Institutes of Health (AM 12705) and the North Dakota Affiliate of the American Diabetes Association. J.B.B. is a recipient of a Research Career Development Award (AM 00428) and his studies were supported by a grant from the National Institutes of Health (AM 28176). A part of the data in this paper was taken from a dissertation submitted by M.A.Y. to the Graduate School of the University of North Dakota. References 1 Veneziale, C.M. (1971) Biochemistry 10, 3443-3447 2 Veneziale, C.M. (1972) Biochemistry 11, 3286-3289 3 Tolbert, M.E.M., Butcher, F.R. and Fain, J.N. (1973) J. Biol. Chem. 248, 5686-5692 4 Garrison, J.C. and Haynes, R.C., Jr. (1973) J. Biol. Chem. 248,5333-5343 5 Tolbert, M.E.M. and Fain, J.N. (1974) J. Biol. Chem. 249, 4462-4466 6 Exton, J.H. and Harper, S.C. (1975) Adv. Cyclic Nucleotide Res. 5,519-532 7 Fain, J.N., Tolbert, M.E.M., Pointer, R.H., Butcher, F.R. and Arnold, A. (1975) Metabolism 24,395-407 8 Feliu, J.E., Hue, L. and Hers, H.B. (1976) Proe. Natl. Acad. Sci. U.S.A. 73, 2762-2766 9 Pilkis, S.J., Riou, J.P. and Claus, T.H. (1976) J. Biol. Chem. 251, 7841-7852 10 Chan, T.M. and Exton, J.H. (1978) J. Biol. Chem. 253, 6393-6400
11 Foster, J.L. and Blair, J.B. (1978) Arch. Biochem. Biophys. 189,263-276 12 Blair, J.B., James, M.E. and Foster, J.L. (1979) J. Biol. Chem. 254, 7585-7590 13 Blair, J.B., James, M.E. and Foster, J.L. (1979) J. Biol. Chem. 254, 7579-7584 14 Rizza, R.A., Cryer, P.E., Haymond, M.W. and Gerich, J.E. (1980) J. Clin. Invest. 65,682-689 15 Hanson, R.W. (1980) Trends Biochem. Sci. 5, l - I I 16 Yorek, M.A., Rufo, G.A., Jr. and Ray, P.D. (1980) Bioclaim. Biophys. Acta 632, 517-526 17 Rufo, G.A., Jr., Yorek, M.A. and Ray, P.D. (1981) Biochim. Biophys. Acta 674,307-315 18 Blair, J.B., Cimbala, M.A., Foster, J.L. and Morgan, R.A. (1976) J. Biol. Chem. 251, 3756-3762 19 Bergmeyer, H.U. (1965) Methods of Enzymatic Analysis, 1st edn., pp. 117-123, Academic Press, New York 20 Exton, J.H. and Park, C.R. (1969) J. Biol. Chem. 244, 1424-1433 21 Blair, J.B., Cook, D.E. and Lardy, H.A. (1973) J. Biol. Chem. 248, 3601-3607 22 Clark, M.G., Kneel N.M., Bosch, A.L. and Lardy, H.A. (1974) J. Biol. Chem. 249, 5695-5703 23 Sutherland, E.W. (1950) Recent Prog. Horm. Res. 5, 441-463 24 Ellis, S. (1956) Pharmacol. Rev. 8,485-562 25 Sutherland, E.W. and RaU, T.W. (1960) Pharmacol. Rev. 12,265-299 26 Sutherland, E.W. and Robison, G.A. (1969) Diabetes 18, 797-819 27 Exton, J.H., Mallette, L.E., Jefferson, L.S., Wong, E.H.A., Friedmann, N. and Park, C.R. (1970) Am. J. Clin. Nutr. 23,993-1003 28 Robison, G.A., Butcher, R.W. and Sutheriand, E.W. (1967) Ann. N.Y. Acad. Sci. 139,703-723 29 Hornbrook, K.R. (1970) Fed. Proc. 29, 1381-1385 30 Kemp, B.E. and Clark, M.G. (1978) J. Biol. Chem. 253, 5147-5154 31 Johnson, M.L. and Veneziale, C.M. (1980) Biochemistry 19, 2191-2195 32 Van Berkel, J.C., Koster, J.F. and Hiilsmann, W.C. (1972) Biochim. Biophys. Acta 276,425-429 33 Van Berkel, J.C., Kruizt, J.K. and Koster, J.F. (1977) Biochim. Biophys. Acta 500,267-276 34 Ishibashi, H. and Cottam, G.L. (1978) J. Biol. Chem. 253, 8767-8771