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Pituitary peptides with direct action on the metabolism of carbohydrates and fatty acids
BIOCHIMICA ET BIOPHYSICA ACTA
3I
BBA 25907 PITUITARY P E P T I D E S W I T H DIRECT ACTION ON T H E METABOLISM OF CARBOHYDRATES AND FATTY ACIDS J. BORNSTEIN, M. E. KRAHL*, L. B. MARSHALL, M. K. GOULD AND J. McD. ARMSTRONG
Department of Biochemistry, Monash University, Clayton, Victoria (Australia) (Received August 7th, 1967)
SUMMARY
An improved method for preparing biologically active polypeptides from the anterior pituitary is described. Two active fractions have been obtained. One fraction accelerates fatty acid synthesis from acetate by liver slices. The other fraction inhibits this synthesis in slices, homogenates and a soluble fraction from liver. This fraction also inhibits the enzymic activity of glyceraldehyde-3-phosphate dehydrogenase and ~-glycerophosphate dehydrogenase. It has no effect on the activity of hexokinase, phosphofructokinase, aldolase, lactate dehydrogenase, glucose6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, malic enzyme or aspartate aminotransferase.
INTRODUCTION
Polypeptide fractions derived from the pituitary glands of various species have been shown to affect glucose uptake and protein synthesis by muscle and fatty acid synthesis by liver 1,2.These fractions were obtained in such small quantities that further studies on their biological and chemical properties were extremely difficult. The present paper describes a simplified method for obtaining these fractions in higher yields. Two fractions have been separated, corresponding to the fractions obtained previously. One fraction accelerates fatty acid synthesis by liver, while the other fraction inhibits this synthesis, and has been shown to inhibit crystalline glyceraldehyde-3-phosphate dehydrogenase and a-glycerophosphate dehydrogenase. MATERIALS AND METHODS
The crystalline enzymes used were obtained from C.F. Boehringer und Soehne, Mannheim, except for liver alcohol dehydrogenase, which was from Sigma Chemical Co., St. Louis, Mo. Sheep pituitary glands were obtained from a local slaughter house and were stored at --2o°. Acetone powders were prepared from the anterior lobes of the glands, and were stored at 4 ° . * Visiting Professor of Biochemistry, 1966. Permanent address: Department of Physiology, University of Chicago, 951 East 58 Street, Chicago 37, Ill. U.S.A.
Biochirn. Biophys. Acta, 156 (I968) 31-37
I. BORNSTEINet al.
32
Dowex 5oW-X2 large pore resin (lOO-2OO mesh) was obtained from Bio-Rad Laboratories. The resin was freed of fines and was cleaned by 5 successive cycles through the sodium and hydrogen forms. Columns were prepared in the hydrogen form and washed with water until the eluate was approx, pH 5. CG5o (200-400 mesh) was obtained from Mallinckrodt Laboratories, and was treated as described by HIRS, MOORE AND STEIN3. Columns were prepared in the hydrogen form and were washed with water until the eluate was approx, pH 5. The biological activity of the various fractions was assayed by measuring the incorporation of ['4C]acetate into fatty acids by liver slices, as described by BORNSTEIN AND HYDE1. The saponifiable lipid samples were dissolved directly in a toluene scintillation fluid (0.5 % 2,5-diphenyloxazole, 0.03 % 1,4-bis-2-(4-methyl-5-phenyloxazolyl)benzene) and counted in a Nuclear Chicago Unilux liquid scintillation counter. Sodium [i-14Clacetate was obtained from the Radiochemical Centre, Amersham, and used at an activity of 1.25/~C/mmole. RESULTS
Preparation of pituitary fractior~s 25 g of acetone-dried pituitary powder were suspended in 5o0 ml of o.I M acetic acid, adjusted to pH 2.2 with 6 M hydrochloric acid and stirred for 5 h at 4 °. The suspension was centrifuged at 40000 x g for 20 min. The residue was stirred for 2 h with IOO ml of o.I M acetic acid and again centrifuged. The clear brown supernatants were combined and ultrafiltered through acid-washed cellophane (British Cellophane Ltd., PT3oo) under 3.2 atm pressure at 4 °. The clear colourless filtrate was applied to a column of Dowex 5oW-X2H, IOO mm x 12 mm, at 4 ° and doveloped successively with 200 ml water, 500 ml triethylamine acetate buffer (0.03 M acetic acid, o.o18 M triethylamine, pH 4.8) and finally with 0.05 M triethylamine. The absorption of the eluate was monitored at 256 m/~. The elution diagram is shown in Fig. I. The middle peak was divided into 3 parts (fractions MPF, MP and MPB), representing the front, middle and back parts of the peak, which were freeze-dried. Samples of each fraction were dissolved in water, adjusted to pH 6.8 with sodium hydroxide and applied to a column of CG 5oH, 60 mm × 5 mm. The column was developed at 20-25 ° with IOO ml water, followed
1.0-
t oJ
"6
MPF I I I I I I
8 o.~c
m
O
.:
Woter
>~Triethylomineaceta;ce pH 4.8
IMp I I I I
MPB I I I I I I
I I I I I I
xTHethylamine >
Fig. I. E l u t i o n p a t t e r n of ultrafiltrate on D o w e x 5 o. Only the peak eluted with triethylamine acetate buffer affected f a t t y acid synthesis. The front and back peaks, eluting with water and triethylamine respectively h a d no effect on f a t t y acid synthesis a n d were discarded.
13iochim. Biophys. Acla, 156 (1968) 31-37
PEPTIDES
AFFECTING
GLUCOSE AND FAT METABOLISM
33
by 15o ml o.o3 M hydrochloric acid, absorption of the eluate being measured at 260 m~. Two subfractions were thus obtained, fraction Ac-P, which was not adsorbed on the resin and which was eluted with water, and fraction In-P which was eluted with 0.03 M HC1. Both fractions were freeze-dried. Fraction MPF yielded mainly fraction Ac-P, fraction MPB yielded mainly fraction In-P, while fraction MP was found to be a mixture of the two. Fractions Ac-P and In-P were ninhydrin positive, and the ninhydrin colour was markedly increased if the fractions were first hydrolysed with 2 M sodium hydroxide. Electrophoresis on paper in pyridine acetate buffer (pH 4.5), showed that fraction Ac-P contained 2 ninhydrin-reacting components and fraction In-P 3 such components. The total middle peak eluted from Dowex 50 columns contained up to 7 ninhydrin-positive components.
Biological activity of the fractions The effect of fractions MP, MPF, MPB, and In-P on the incorporation of acetate into f a t t y acids is shown in Table I. Fraction MPF accelerated fatty acid synthesis while fractions MP and MPB were inhibitory. Separation into fractions Ac-P and In-P resulted in a more marked inhibition by fraction In-P. Fractions prepared from TABLE
I
EFFECT OF FRACTIONS M P F , BY LIVER SLICES
MP,
MPB
All fractions were tested at an amount
Expt. No.
AND I n - P
ON FATTY ACID SYNTHESIS FROM ACETATE
e q u i v a l e n t t o i o o m g of p i t u i t a r y p o w d e r .
Additions
Counts/rain per g liver
Change (%)
o MPF MP MPB
83 138 31 14
500 ooo 600 500
+65 --62 --83
o In-P
872 ooo 5° 60o
--94
14 extracts have been tested, and all showed similar effects. Fig. 2 shows that the inhibitory effect of fraction MPB was concentration dependent, and that the reciprocal of the inhibition was proportional to the log dose of fraction MPB over 3 orders of magnitude. Experiments were then carried out to determine whether the inhibition of f a t t y acid synthesis by fractions MPB and In-P could be demonstrated in broken cell systems. Three systems were investigated. (I) A crude liver homogenate in o.I M phosphate buffer (pH 6.8), incubated with Io mM potassium citrate and 0.3 mM sodium acetate. (2) and (3) The whole homogenate and soluble system prepared and assayed according to M A S O R O , K O R C H A K AND PORTER 4. The results (Table II) clearly show that inhibition was obtained in all three systems. Biochim. Biophys. Acta, 1 5 6 (1968) 3 1 - 3 7
J. BORNSTEIN et
34
al.
Preliminary experiments on the concentrations of glycolytic intermediates in liver slices and homogenates, incubated with fraction MPB, suggested that inhibition of glucose utilisation occurred at the triose phosphate level.
/3 .c
.c_
tog (dose)
Fig. 2. D o s e - r e s p o n s e c u r v e for t h e i n h i b i t i o n b y f r a c t i o n M P B of f a t t y a c i d s y n t h e s i s from a c e t a t e b y l i v e r slices. The a b s c i s s a is in a r b i t r a r y units, w i t h t h e l o w e s t v a l u e (log (dose) = i) corres p o n d i n g to a p p r o x . 2 o o / , g of p i t u i t a r y powder.
TABLE II INHIBITION OF FATTY ACID SYNTHESIS IN CELL-FREE SYSTEMS BY FRACTIONS M P B AND I n - P The s y s t e m s used are d e s c r i b e d in t h e t e x t . E x t r a c t i o w a s t e s t e d a t t h e e q u i v a l e n t of IOO m g p i t u i t a r y powd er, a n d e x t r a c t I7 a t t h e e q u i v a l e n t of 75 m g p i t u i t a r y pow de r.
System
Extract
Addition
Counts/rain per g liver
Inhibition (%)
Phosphate homogenate
io
o MPB In-P
2o 4o0 6 ooo 4 ooo
7z 80
O MPB
31 OOO 9 900
68
710 235
67
H o m o g e n a t e (MASORO, KORCHAK
17
AND PORTER 4) Soluble (MASORO, KORCHAK AND PORTER 4)
17
O MPB
TABLE III INHIBITION OF GLYCERALDEHYDE-3-PHOSPHATE DEtfYDROGENASE ACTIVITY The e n z y m e a c t i v i t y was m e a s u r e d as d e s c r i b e d b y PIHL AND LANGE 5. I n h i b i t i o n s were c a l c u l a t e d as d e s c r i b e d b y BORNSTEIN, ARMSTRONG AND JONES 6.
Fraction added
Equivalent weight of Inhibition pituitary powder (%)
MPB In-P
a p p r o x . 200 m g approx, ioo mg
41 48
Biochim. Biophys. Acta, 156 (1968) 31-37
PEPTIDES AFFECTING GLUCOSE AND FAT METABOLISM
35
We therefore investigated the effect of fractions MPB and In-P on the activity of crystalline glyceraldehyde-3-phosphate dehydrogenase. As shown in Table III, both fractions inhibited glyceraldehyde-3-phosphate dehydrogenase. The effect of fraction In-P on the activity of a number of mammalian enzymes was tested. Only glyceraldehyde-3-phosphate dehydrogenase and a-glycerophosphate dehydrogenase were affected (Table IV). It has been found that solutions of fraction MPB remained active for several weeks when stored frozen. However, solutions of fraction In-P were inactivated by freezing although activity was retained for several days in 5 mM acetic acid at 4 °. TABLE IV EFFECT
OF
FRACTION
ID-P
ON
THE
ACTIVITY OF
VARIOUS
ENZYMES
The m e t h o d used for the assay of each enzyme is given b y the reference indicated. The r a t liver e x t r a c t was the s u p e r n a t a n t o b t a i n e d after centrifuging an h o m o g e n a t e at i o o o o o x g for 30 min. The a m o u n t of fraction I n - P used in each assay corresponded to 8oo m g of p i t u i t a r y p o w d e r unless otherwise indicated.
Enzyme
Source
Hexokinase 7 Aldolase a P h o s p h o f r u c t o k i n a s ea Lactate dehydrogenase a
a - G l y c e r o p h o s p h a t e dehydrogenase 9
R a t muscle* R a t liver e x t r a c t R a t liver e x t r a c t R a b b i t muscle, crystalline R a t liver e x t r a c t R a t liver e x t r a c t R a t liver e x t r a c t R a t liver e x t r a c t Horse liver, crystalline R a t liver e x t r a c t R a t liver e x t r a c t H u m a n serum R a b b i t muscle, crystalline
Glyceraldehyde-3-phosphate dehydrogenase b
R a b b i t muscle, crystalline
Glucose-6-phosphate dehydrogenase 9 Malate dehydrogenase 9 Isocitrate dehydrogenase 1° Alcohol dehydrogenase n Malic enzyme 12 A s p a r t a t e a m i n o t r a n s f e r a s e 13
Inhibition (%) o o o o o o o o o o o o IOO 70 49 i8"*
* Partially purified according to CRANE AND SOLS7, ** F r a c t i o n I n - P equivalent to 8o mg of p i t u i t a r y powder.
DISCUSSION
The experiments reported here confirm the earlier findings of BORNSTEINet al.1, ~ of the effects of anterior pituitary extracts on glucose utilisation and on fatty acid synthesis by liver. The present method for the preparation of the active fractions has several advantages over the earlier procedure, being simpler and giving greater yields, and providing a complete separation of the acceleratory (Ac-P) and inhibitory (In-P) activities. Both fractions Ac-P and In-P contain polypeptide material, but neither fraction was homogeneous on electrophoresis. The preparative procedure relies on frontal elution chromatography, and gives a considerable purification of the ultrafiltrate of pituitary extract. So far we have been unable to obtain separation of the components of fractions Ac-P and In-P by Biochim. Biophys. Acta, 156 (1968) 31-37
36
j. BORNSTEIN et al.
chromatography on buffered ion exchange columns or by gel filtration, and further progress is complicated by the instability of these fractions. Incorporation of acetate into fatty acids by the cytoplasmic system represents de novo synthesis of fatty acids, whereas the particulate system incorporates acetate principally by the elongation of existing fatty acid chains 14. Thus it can be seen that fraction In-P inhibits de novo fatty acid synthesis rather than chain elongation, since it acts on the soluble system. The effect of fraction In-P on glucose metabolism can be explained by the finding that this fraction inhibits both glyceraldehyde-3-phosphate dehydrogenase and NADdependent a-glycerophosphate dehydrogenase. The inhibition of glyceraldehyde phosphate dehydrogenase, and the consequent inhibition of glycolysis could explain the diabetogenic activity of anterior pituitary extracts, which was first observed by Y O U N G 15.
The multiple inhibitory actions of fraction In-P pose some difficulties. Since the fraction is not homogeneous, it cannot be stated whether or not glyceraldehyde-3phosphate dehydrogenase and c~-glycerophosphate dehydrogenase are inhibited by the same component. Since both enzymes are NAD-dependent and act on isomers of triose phosphate, it is not inconceivable that the same component would inhibit both enzymes. The inhibition of f a t t y acid synthesis by fraction In-P could suggest that there is yet another active component in this fraction, distinct from the component(s) acting on the triose phosphate dehydrogenases. However, it is possible to explain the inhibition of fatty acid synthesis as a consequence of the inhibition of c~-glycerophosphate dehydrogenase. BORTZ AND LYNENTM showed that liver acetyl-CoA carboxylase is strongly inhibited by long chain acyl-CoA compounds. It has also been shown that fatty acid synthesis in cell-free preparations is stimulated by a-glycerophosphate, and this is attributed to the removal of palmityl-CoA by glyceride formation 17. Therefore limitation of a-glycerophosphate formation by the inhibition of c~-glycerophosphate dehydrogenase would decrease glyceride formation, and the resulting accumulation of palmityl, CoA would inhibit further fatty acid synthesis. While this explanation is satisfactory for liver slices and homogenates, in the soluble system microsomes must be present for glyceride synthesis to occur. However, HOWARD AND LOWENSTEIN17 showed that the amount of microsomal material required for glyceride formation is extremely small. Thus a small contamination of the soluble system (Table II) would permit glyceride synthesis to occur. ACKNOWLEDGEMENTS
This work was supported in part by the National Health and Medical Research Council of Australia and by Merck, Sharp and Dohme (Australia). We wish to thank William Angliss and Co. (Australia) Pty. Ltd., for their helpfulness in collecting the pituitary glands used in this work. REFERENCES I J. BORNSTEIN AND D. HYDE, Nature, 187 (196o) 125. 2 J. BORNSTIgIN, D. HYDE AND K. J. CATT,Ciba Foundation Colloquia on Endocrinology, Vol. 15, Churchill, L o n d o n , 1964, p. 240.
Biochim. Biophys. Acta, 156 (1968) 31-37
PEPTIDES AFFECTING GLUCOSE AND FAT METABOLISM 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17
37
C. H. -W. HIES, S. MOORE AND W. H. STEIN, J. Biol. Chem., 200 (1953) 493. E. J. MASORO, H. M. KORCHAK AND E. PORTER, Biochim. Biophys. Acta, 58 (1962) 4o7: A. PIHL AND R. LANGE, J. Biol. Chem., 237 (1962) 1356. J. BORNSTEIN, J. McD. ARMSTRONG AND M. D. JoNEs, Biochim. Biophys. Acta, 156 (1968) 38. R. K. CRANE AND A. SOLS, in S. P. COLOWICK AND ~ . O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 277. J. A. SIBLEY AND A. L. LEHNINGER, J. Biol. Chem., i77 (1949) 859. C. E. SHONK AND G. E. BOXER, Cancer Res., 24 (I964) 709 . S. K. ~VoLFSON AND H. G. WILLIAMS-ASHMAN, Proc. Soc. Exptl. Biol. iVied., 96 (1957) 231. H. THEORELL AND R. I(. BONNICHSEN, Acta Chem. Scand., 5 (1951) 11o5. S. OCHOA, in S. P. COLOWlCK ANn N. O. t(APLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 739S. REITMAN AND S. FRANKEL, Am. jr. Clin. Pathol., 28 (1957) 56. J. A. OLSON, Ann. Rev. Biochem., 35 (1966) 563. V. G. YOUNG, Lancet, 233 (1937) 372. W. M. BORTZ AND F. LYNEN, Biochem. Z., 337 (1963) 505 • C. F. HOWARD AND J. M. LOWENSTEIN, J. Biol. Chem., 240 (1965) 417 o.
Biochim. Biophys. Acta, 156 (1968) 31-37
3I
BBA 25907 PITUITARY P E P T I D E S W I T H DIRECT ACTION ON T H E METABOLISM OF CARBOHYDRATES AND FATTY ACIDS J. BORNSTEIN, M. E. KRAHL*, L. B. MARSHALL, M. K. GOULD AND J. McD. ARMSTRONG
Department of Biochemistry, Monash University, Clayton, Victoria (Australia) (Received August 7th, 1967)
SUMMARY
An improved method for preparing biologically active polypeptides from the anterior pituitary is described. Two active fractions have been obtained. One fraction accelerates fatty acid synthesis from acetate by liver slices. The other fraction inhibits this synthesis in slices, homogenates and a soluble fraction from liver. This fraction also inhibits the enzymic activity of glyceraldehyde-3-phosphate dehydrogenase and ~-glycerophosphate dehydrogenase. It has no effect on the activity of hexokinase, phosphofructokinase, aldolase, lactate dehydrogenase, glucose6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, malic enzyme or aspartate aminotransferase.
INTRODUCTION
Polypeptide fractions derived from the pituitary glands of various species have been shown to affect glucose uptake and protein synthesis by muscle and fatty acid synthesis by liver 1,2.These fractions were obtained in such small quantities that further studies on their biological and chemical properties were extremely difficult. The present paper describes a simplified method for obtaining these fractions in higher yields. Two fractions have been separated, corresponding to the fractions obtained previously. One fraction accelerates fatty acid synthesis by liver, while the other fraction inhibits this synthesis, and has been shown to inhibit crystalline glyceraldehyde-3-phosphate dehydrogenase and a-glycerophosphate dehydrogenase. MATERIALS AND METHODS
The crystalline enzymes used were obtained from C.F. Boehringer und Soehne, Mannheim, except for liver alcohol dehydrogenase, which was from Sigma Chemical Co., St. Louis, Mo. Sheep pituitary glands were obtained from a local slaughter house and were stored at --2o°. Acetone powders were prepared from the anterior lobes of the glands, and were stored at 4 ° . * Visiting Professor of Biochemistry, 1966. Permanent address: Department of Physiology, University of Chicago, 951 East 58 Street, Chicago 37, Ill. U.S.A.
Biochirn. Biophys. Acta, 156 (I968) 31-37
I. BORNSTEINet al.
32
Dowex 5oW-X2 large pore resin (lOO-2OO mesh) was obtained from Bio-Rad Laboratories. The resin was freed of fines and was cleaned by 5 successive cycles through the sodium and hydrogen forms. Columns were prepared in the hydrogen form and washed with water until the eluate was approx, pH 5. CG5o (200-400 mesh) was obtained from Mallinckrodt Laboratories, and was treated as described by HIRS, MOORE AND STEIN3. Columns were prepared in the hydrogen form and were washed with water until the eluate was approx, pH 5. The biological activity of the various fractions was assayed by measuring the incorporation of ['4C]acetate into fatty acids by liver slices, as described by BORNSTEIN AND HYDE1. The saponifiable lipid samples were dissolved directly in a toluene scintillation fluid (0.5 % 2,5-diphenyloxazole, 0.03 % 1,4-bis-2-(4-methyl-5-phenyloxazolyl)benzene) and counted in a Nuclear Chicago Unilux liquid scintillation counter. Sodium [i-14Clacetate was obtained from the Radiochemical Centre, Amersham, and used at an activity of 1.25/~C/mmole. RESULTS
Preparation of pituitary fractior~s 25 g of acetone-dried pituitary powder were suspended in 5o0 ml of o.I M acetic acid, adjusted to pH 2.2 with 6 M hydrochloric acid and stirred for 5 h at 4 °. The suspension was centrifuged at 40000 x g for 20 min. The residue was stirred for 2 h with IOO ml of o.I M acetic acid and again centrifuged. The clear brown supernatants were combined and ultrafiltered through acid-washed cellophane (British Cellophane Ltd., PT3oo) under 3.2 atm pressure at 4 °. The clear colourless filtrate was applied to a column of Dowex 5oW-X2H, IOO mm x 12 mm, at 4 ° and doveloped successively with 200 ml water, 500 ml triethylamine acetate buffer (0.03 M acetic acid, o.o18 M triethylamine, pH 4.8) and finally with 0.05 M triethylamine. The absorption of the eluate was monitored at 256 m/~. The elution diagram is shown in Fig. I. The middle peak was divided into 3 parts (fractions MPF, MP and MPB), representing the front, middle and back parts of the peak, which were freeze-dried. Samples of each fraction were dissolved in water, adjusted to pH 6.8 with sodium hydroxide and applied to a column of CG 5oH, 60 mm × 5 mm. The column was developed at 20-25 ° with IOO ml water, followed
1.0-
t oJ
"6
MPF I I I I I I
8 o.~c
m
O
.:
Woter
>~Triethylomineaceta;ce pH 4.8
IMp I I I I
MPB I I I I I I
I I I I I I
xTHethylamine >
Fig. I. E l u t i o n p a t t e r n of ultrafiltrate on D o w e x 5 o. Only the peak eluted with triethylamine acetate buffer affected f a t t y acid synthesis. The front and back peaks, eluting with water and triethylamine respectively h a d no effect on f a t t y acid synthesis a n d were discarded.
13iochim. Biophys. Acla, 156 (1968) 31-37
PEPTIDES
AFFECTING
GLUCOSE AND FAT METABOLISM
33
by 15o ml o.o3 M hydrochloric acid, absorption of the eluate being measured at 260 m~. Two subfractions were thus obtained, fraction Ac-P, which was not adsorbed on the resin and which was eluted with water, and fraction In-P which was eluted with 0.03 M HC1. Both fractions were freeze-dried. Fraction MPF yielded mainly fraction Ac-P, fraction MPB yielded mainly fraction In-P, while fraction MP was found to be a mixture of the two. Fractions Ac-P and In-P were ninhydrin positive, and the ninhydrin colour was markedly increased if the fractions were first hydrolysed with 2 M sodium hydroxide. Electrophoresis on paper in pyridine acetate buffer (pH 4.5), showed that fraction Ac-P contained 2 ninhydrin-reacting components and fraction In-P 3 such components. The total middle peak eluted from Dowex 50 columns contained up to 7 ninhydrin-positive components.
Biological activity of the fractions The effect of fractions MP, MPF, MPB, and In-P on the incorporation of acetate into f a t t y acids is shown in Table I. Fraction MPF accelerated fatty acid synthesis while fractions MP and MPB were inhibitory. Separation into fractions Ac-P and In-P resulted in a more marked inhibition by fraction In-P. Fractions prepared from TABLE
I
EFFECT OF FRACTIONS M P F , BY LIVER SLICES
MP,
MPB
All fractions were tested at an amount
Expt. No.
AND I n - P
ON FATTY ACID SYNTHESIS FROM ACETATE
e q u i v a l e n t t o i o o m g of p i t u i t a r y p o w d e r .
Additions
Counts/rain per g liver
Change (%)
o MPF MP MPB
83 138 31 14
500 ooo 600 500
+65 --62 --83
o In-P
872 ooo 5° 60o
--94
14 extracts have been tested, and all showed similar effects. Fig. 2 shows that the inhibitory effect of fraction MPB was concentration dependent, and that the reciprocal of the inhibition was proportional to the log dose of fraction MPB over 3 orders of magnitude. Experiments were then carried out to determine whether the inhibition of f a t t y acid synthesis by fractions MPB and In-P could be demonstrated in broken cell systems. Three systems were investigated. (I) A crude liver homogenate in o.I M phosphate buffer (pH 6.8), incubated with Io mM potassium citrate and 0.3 mM sodium acetate. (2) and (3) The whole homogenate and soluble system prepared and assayed according to M A S O R O , K O R C H A K AND PORTER 4. The results (Table II) clearly show that inhibition was obtained in all three systems. Biochim. Biophys. Acta, 1 5 6 (1968) 3 1 - 3 7
J. BORNSTEIN et
34
al.
Preliminary experiments on the concentrations of glycolytic intermediates in liver slices and homogenates, incubated with fraction MPB, suggested that inhibition of glucose utilisation occurred at the triose phosphate level.
/3 .c
.c_
tog (dose)
Fig. 2. D o s e - r e s p o n s e c u r v e for t h e i n h i b i t i o n b y f r a c t i o n M P B of f a t t y a c i d s y n t h e s i s from a c e t a t e b y l i v e r slices. The a b s c i s s a is in a r b i t r a r y units, w i t h t h e l o w e s t v a l u e (log (dose) = i) corres p o n d i n g to a p p r o x . 2 o o / , g of p i t u i t a r y powder.
TABLE II INHIBITION OF FATTY ACID SYNTHESIS IN CELL-FREE SYSTEMS BY FRACTIONS M P B AND I n - P The s y s t e m s used are d e s c r i b e d in t h e t e x t . E x t r a c t i o w a s t e s t e d a t t h e e q u i v a l e n t of IOO m g p i t u i t a r y powd er, a n d e x t r a c t I7 a t t h e e q u i v a l e n t of 75 m g p i t u i t a r y pow de r.
System
Extract
Addition
Counts/rain per g liver
Inhibition (%)
Phosphate homogenate
io
o MPB In-P
2o 4o0 6 ooo 4 ooo
7z 80
O MPB
31 OOO 9 900
68
710 235
67
H o m o g e n a t e (MASORO, KORCHAK
17
AND PORTER 4) Soluble (MASORO, KORCHAK AND PORTER 4)
17
O MPB
TABLE III INHIBITION OF GLYCERALDEHYDE-3-PHOSPHATE DEtfYDROGENASE ACTIVITY The e n z y m e a c t i v i t y was m e a s u r e d as d e s c r i b e d b y PIHL AND LANGE 5. I n h i b i t i o n s were c a l c u l a t e d as d e s c r i b e d b y BORNSTEIN, ARMSTRONG AND JONES 6.
Fraction added
Equivalent weight of Inhibition pituitary powder (%)
MPB In-P
a p p r o x . 200 m g approx, ioo mg
41 48
Biochim. Biophys. Acta, 156 (1968) 31-37
PEPTIDES AFFECTING GLUCOSE AND FAT METABOLISM
35
We therefore investigated the effect of fractions MPB and In-P on the activity of crystalline glyceraldehyde-3-phosphate dehydrogenase. As shown in Table III, both fractions inhibited glyceraldehyde-3-phosphate dehydrogenase. The effect of fraction In-P on the activity of a number of mammalian enzymes was tested. Only glyceraldehyde-3-phosphate dehydrogenase and a-glycerophosphate dehydrogenase were affected (Table IV). It has been found that solutions of fraction MPB remained active for several weeks when stored frozen. However, solutions of fraction In-P were inactivated by freezing although activity was retained for several days in 5 mM acetic acid at 4 °. TABLE IV EFFECT
OF
FRACTION
ID-P
ON
THE
ACTIVITY OF
VARIOUS
ENZYMES
The m e t h o d used for the assay of each enzyme is given b y the reference indicated. The r a t liver e x t r a c t was the s u p e r n a t a n t o b t a i n e d after centrifuging an h o m o g e n a t e at i o o o o o x g for 30 min. The a m o u n t of fraction I n - P used in each assay corresponded to 8oo m g of p i t u i t a r y p o w d e r unless otherwise indicated.
Enzyme
Source
Hexokinase 7 Aldolase a P h o s p h o f r u c t o k i n a s ea Lactate dehydrogenase a
a - G l y c e r o p h o s p h a t e dehydrogenase 9
R a t muscle* R a t liver e x t r a c t R a t liver e x t r a c t R a b b i t muscle, crystalline R a t liver e x t r a c t R a t liver e x t r a c t R a t liver e x t r a c t R a t liver e x t r a c t Horse liver, crystalline R a t liver e x t r a c t R a t liver e x t r a c t H u m a n serum R a b b i t muscle, crystalline
Glyceraldehyde-3-phosphate dehydrogenase b
R a b b i t muscle, crystalline
Glucose-6-phosphate dehydrogenase 9 Malate dehydrogenase 9 Isocitrate dehydrogenase 1° Alcohol dehydrogenase n Malic enzyme 12 A s p a r t a t e a m i n o t r a n s f e r a s e 13
Inhibition (%) o o o o o o o o o o o o IOO 70 49 i8"*
* Partially purified according to CRANE AND SOLS7, ** F r a c t i o n I n - P equivalent to 8o mg of p i t u i t a r y powder.
DISCUSSION
The experiments reported here confirm the earlier findings of BORNSTEINet al.1, ~ of the effects of anterior pituitary extracts on glucose utilisation and on fatty acid synthesis by liver. The present method for the preparation of the active fractions has several advantages over the earlier procedure, being simpler and giving greater yields, and providing a complete separation of the acceleratory (Ac-P) and inhibitory (In-P) activities. Both fractions Ac-P and In-P contain polypeptide material, but neither fraction was homogeneous on electrophoresis. The preparative procedure relies on frontal elution chromatography, and gives a considerable purification of the ultrafiltrate of pituitary extract. So far we have been unable to obtain separation of the components of fractions Ac-P and In-P by Biochim. Biophys. Acta, 156 (1968) 31-37
36
j. BORNSTEIN et al.
chromatography on buffered ion exchange columns or by gel filtration, and further progress is complicated by the instability of these fractions. Incorporation of acetate into fatty acids by the cytoplasmic system represents de novo synthesis of fatty acids, whereas the particulate system incorporates acetate principally by the elongation of existing fatty acid chains 14. Thus it can be seen that fraction In-P inhibits de novo fatty acid synthesis rather than chain elongation, since it acts on the soluble system. The effect of fraction In-P on glucose metabolism can be explained by the finding that this fraction inhibits both glyceraldehyde-3-phosphate dehydrogenase and NADdependent a-glycerophosphate dehydrogenase. The inhibition of glyceraldehyde phosphate dehydrogenase, and the consequent inhibition of glycolysis could explain the diabetogenic activity of anterior pituitary extracts, which was first observed by Y O U N G 15.
The multiple inhibitory actions of fraction In-P pose some difficulties. Since the fraction is not homogeneous, it cannot be stated whether or not glyceraldehyde-3phosphate dehydrogenase and c~-glycerophosphate dehydrogenase are inhibited by the same component. Since both enzymes are NAD-dependent and act on isomers of triose phosphate, it is not inconceivable that the same component would inhibit both enzymes. The inhibition of f a t t y acid synthesis by fraction In-P could suggest that there is yet another active component in this fraction, distinct from the component(s) acting on the triose phosphate dehydrogenases. However, it is possible to explain the inhibition of fatty acid synthesis as a consequence of the inhibition of c~-glycerophosphate dehydrogenase. BORTZ AND LYNENTM showed that liver acetyl-CoA carboxylase is strongly inhibited by long chain acyl-CoA compounds. It has also been shown that fatty acid synthesis in cell-free preparations is stimulated by a-glycerophosphate, and this is attributed to the removal of palmityl-CoA by glyceride formation 17. Therefore limitation of a-glycerophosphate formation by the inhibition of c~-glycerophosphate dehydrogenase would decrease glyceride formation, and the resulting accumulation of palmityl, CoA would inhibit further fatty acid synthesis. While this explanation is satisfactory for liver slices and homogenates, in the soluble system microsomes must be present for glyceride synthesis to occur. However, HOWARD AND LOWENSTEIN17 showed that the amount of microsomal material required for glyceride formation is extremely small. Thus a small contamination of the soluble system (Table II) would permit glyceride synthesis to occur. ACKNOWLEDGEMENTS
This work was supported in part by the National Health and Medical Research Council of Australia and by Merck, Sharp and Dohme (Australia). We wish to thank William Angliss and Co. (Australia) Pty. Ltd., for their helpfulness in collecting the pituitary glands used in this work. REFERENCES I J. BORNSTEIN AND D. HYDE, Nature, 187 (196o) 125. 2 J. BORNSTIgIN, D. HYDE AND K. J. CATT,Ciba Foundation Colloquia on Endocrinology, Vol. 15, Churchill, L o n d o n , 1964, p. 240.
Biochim. Biophys. Acta, 156 (1968) 31-37
PEPTIDES AFFECTING GLUCOSE AND FAT METABOLISM 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17
37
C. H. -W. HIES, S. MOORE AND W. H. STEIN, J. Biol. Chem., 200 (1953) 493. E. J. MASORO, H. M. KORCHAK AND E. PORTER, Biochim. Biophys. Acta, 58 (1962) 4o7: A. PIHL AND R. LANGE, J. Biol. Chem., 237 (1962) 1356. J. BORNSTEIN, J. McD. ARMSTRONG AND M. D. JoNEs, Biochim. Biophys. Acta, 156 (1968) 38. R. K. CRANE AND A. SOLS, in S. P. COLOWICK AND ~ . O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 277. J. A. SIBLEY AND A. L. LEHNINGER, J. Biol. Chem., i77 (1949) 859. C. E. SHONK AND G. E. BOXER, Cancer Res., 24 (I964) 709 . S. K. ~VoLFSON AND H. G. WILLIAMS-ASHMAN, Proc. Soc. Exptl. Biol. iVied., 96 (1957) 231. H. THEORELL AND R. I(. BONNICHSEN, Acta Chem. Scand., 5 (1951) 11o5. S. OCHOA, in S. P. COLOWlCK ANn N. O. t(APLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 739S. REITMAN AND S. FRANKEL, Am. jr. Clin. Pathol., 28 (1957) 56. J. A. OLSON, Ann. Rev. Biochem., 35 (1966) 563. V. G. YOUNG, Lancet, 233 (1937) 372. W. M. BORTZ AND F. LYNEN, Biochem. Z., 337 (1963) 505 • C. F. HOWARD AND J. M. LOWENSTEIN, J. Biol. Chem., 240 (1965) 417 o.
Biochim. Biophys. Acta, 156 (1968) 31-37