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The distribution of some enzymes of glycolysis, gluconeogenesis and lipogenesis in the kidney during development of the rat
Int. J. Biochem., 1975, Vol. 6, pp. 159 to 163. Pergamon Press. Printed in Great Britain
THE DISTRIBUTION
OF SOME ENZYMES
GLUCONEOGENESIS DURING
AND LIPOGENESIS DEVELOPMENT
CHRISTINE
A. HAUSER
AND
‘59
OF GLYCOLYSIS, IN THE KIDNEY
OF THE RAT ERNEST
BAILEY
Department of Biochemistry, The University of Sheffield, Sheffield, SIO 2TN, U.K. (Received 19 October 1974) ABSTRACT I. Throughout development the activities of the glycolytic enzymes hexokinase, phosphofructokinase and pyruvate kinase were much greater in the renal medulla than the renal cortex. 2. In contrast, at all ages studied, the activities of the extramitochondrial enzymes aconitate hydratase, malate dehydrogenase and isocitrate dehydrogenase and the gluconeogenic enzymes, fructose x,6-diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase were greater in the cortex than the medulla. 3. Lipogenesis measured by acetate conversion to lipid and the activities of ‘ malic enzyme ‘, glucose 6-phosphate dehydrogenase and ATP citrate lyase were similar in cortex and medulla at various ages. DETAILED developmental patterns for the activities of rat kidney gluconeogenic, glycolytic and lipogenic enzymes have recently been described (Hauser & Bailey, 1974 a, b). However, the enzyme activities were determined in preparations of whole kidneys. Since gluconeogenesis (Krebs, I 964) and some of the enzymes of gluconeogenesis (Waldman & Burch, 1963; Szepesi, Avery & Freedland, 1970) appear to be located in the kidney cortex in the adult animal it was thought necessary to study the distribution of the enzymes of glycolysis, lipogenesis and gluconeogenesis between the kidney cortex and medulla at various stages of development.
MATERIALS
AND METHODS
MATERIALS
The materials used were obtained from the sources described by Hauser & Bailey (I 974 a, b). ANIMALS AND DIET
Details are as described by Hauser & Bailey (1974 a, b) except that only female rats were used. Adult rats weighed about 200 g. MEASUREMENT
OF ENZYME ACTIVITIJIS
The rats were killed by a blow on the head and cervical fracture, between g a.m. and II a.m.
The kidneys were extracted and the capsules removed. The kidney was then dissected into two, transversely, to expose the different regions. Thin slices were cut from the surface of the cortex to give an uncontaminated cortical preparation. A rough dissection was then made along the corticomedullary junction using a fine scalpel and a dissecting magnifying glass. The medulla and papilla appeared to be relatively free of cortical material. Different homogenates of the cortex and medulla samples were prepared for the various enzymes assayed. Homogenization was carried out using a motor-driven glass-Teflon Potter-Elvehjem homogenizer. For the determination of the activity of ‘ malic enzyme ’ (L-malate : NADP oxidoreductase [decarboxylating], EC I. I. I .40), pyruvate kinase (ATP : pyruvate phosphotransferase, EC 2.7. I .40), phosphofructokinase (ATP : n-fructose 6-phosphate I-phosphotransferase, EC 2.7. I. I I), ATP citrate lyase (ATP-citrate oxaloacetate-lyase [CoA acetylating and ATP dephosphorylating], EC 4. I .3.8) and glucose 6-phosphate dehydrogenase (n-glucose 6-phosphate : NADP oxidoreductase, EC I. I. 1.49) tissue homogenates were prepared as described by Lockwood, Bailey & Taylor (x970). For extramitochondrial NADP-dependent isocitrate dehydrogenase (nL-isocitrate : NADP oxidoreductase [decarboxylating], EC I. I. I .42), extramitochondrial malate dehydrogenase (Lmalate : NAD oxidoreductase, EC 1.x.1.37), extramitochondrial aconitate hydratase (citrate [isocitrate] hydrolyase, EC 4.2. I .3), hexokinase (ATP : n-hexose 6_phosphotransferase, EC 2.7. I. I),
8.97 _+0.6 3.48 + 0.4 0.27 If:0.02 3’8+_0.3 2.5 f 0.2 8.9 + 0.5 430f 14 0~15+0~01 0.45 + 0’02 1’0 + 0.04 Ig’o+o’ooI
7.01 + 0.4 1’19 + 0.04 0’ I + 0.03 2.6g+o.I 1*4+0*04
0’ 144 f 0,007 0’2 +_0’01 I’2 + 0.05 16.7 + 0.002
5;2;:;3
1*4+-0.03 1*4+0.1 17’7+ I
20 days
1*3+0*1 1*0+0*2 182+ I
IO days
CORTEX
0*18_+0*02 1.8ko.2 1.6 + 0.02 I 7.2 +_0.002
$4~~‘;’ . . 16.9 z I 5g5+16
8.51_+ 0.4 3’20+0’3 0.4 1 + 0.05 1.24+0*1
1.63fo.1 1.6ko.2 14’752
Adult
0.157 + 0.003 0’2 + 0’02 1.3 + 0.03 ‘3’4+0’01
1’1 kO.03 0.8 f. 0.04 3.6 + 0.2 17554
4’10+-0.4 0.53 + 0.07 0.05 + 0’0 I
3*0+_0.1 1*5*0.1 29.0 f 3
IO days
0~15+0~01 0.40 f 0’02 1.7 _+0.02 17’0 + 0’002
4.76 + 0.07 255 rt IO
1*0+_0.1
I .06 _+0.03
0.07 _+0’0 I
I’oI+o*I
3’27+0’1
2’1+0*1 2’2 + 0’2 2g*6+3
20 days
MEDULLA
0’20 + 0.007 1.8+0*3 I’5 * 0’02 13’gfo.ooI
3’53ZkO.5 0.58 f 0.04 0.09 + 0.008 0.32 + 0.04 1*1g_+0.1 1’1 kO.07 7’2fo.7 343+5
1.5fo.1 3’4kO.3 34’4 f 3
Adult
The results are the means +_SEM of four determinations. Enzyme activities are expressed as pmoles substrate utilized/minute/g wet wt. of tissue. Acetate incorporation is expressed as nmoles [14C]acetate incorporated into lipid/minute/g. wet wt. of tissue.
GLYCOLYSI~ Hexokinase Phosphofructokinase Pyruvate kinase GLUCONEOGENE~I~ Fructose I ,6_diphosphatase Extramitochondrial PEP carboxykinase lviitochondrial PEP carboxykinase Extramitochondrial pyruvate carboxylase Mitochondrial pyruvate carboxylase Extramitochondrial aconitate hydratase Extramitochondrial isocitrate dehydrogenase Extramitochondrial malate dehydrogenase LIPOGENRSIS ATP citrate lyase ‘ Malic enzyme ’ Glucose 6-phosphate dehydrogenase Acetate incorporation into lipid
ENZYME
Table I.-DISTRIBUTION OF ENZYMEAC-~I~ITYIN THE KIDNEY DURINGDEVELOPMENT
Le
$
B
Isem
KIDNEY ENZYMES DURING DEVELOPMENT
acetate incorporation into lipid, tissue homogenates were prepared as described by Hauser & Bailey (1974 a). For fructose 1,6diphosphatase (n-fructose t,6-diphosphate Iphosphohydrolase, EC 3. I .7. I I), phosphoenolpyruvate carboxykinase (GTP : oxaloacetate carboxylase [transphosphorylating], EC 4. I. I .32), and pyruvate carboxylase (pyruvate : carbon dioxide ligase [ADP], EC 6.4. I. I ) , tissue homogenates were prepared as described by Hauser & Bailey (1974 b). In the case of fructose 1,6and
diphosphatase, phosphoenolpyruvate carboxytissue kinase and pyruvate carboxylase, homogenates were fractionated as described by Hauser & Bailey (I 974 b) . For all other enzymes, tissue homogenates were fractionated as described by Hauser & Bailey (1974 a). Fructose r,6-diphosphatase was assayed by the method of Opie & Newsholme (1967). Phosphopyruvate enolpyruvate carboxykinase and carboxylase were assayed by the methods of Ballard & Hanson (1967). Glucose 6-phosphate dehydrogenase, ATP citrate lyase, pyruvate kinase and ‘ malic enzyme ’ were assayed as described by Taylor, Bailey & Bartley (1967). Phosphofructokinase was assayed as described by Underwood & Newsholme (1965). NADPdependent isocitrate dehydrogenase was assayed by the method of Kornberg (1955) and extramitochondrial aconitate hydratase was assayed by the same method except that I mM trisodium citrate was used as substrate instead of DLisocitric acid. Hexokinase was assaved bv the method of Walker & Holland (1965). kxtramitochondrial malate dehydrogenase was assayed as described by Ochoa (1955). All enzyme assays were carried out at 25” C. [U-%]Acetate incorporation into total lipid was measured at 37” C by a modification of the method of Smith & Dils (1966) as described by Hauser & Bailey (I974
a).
RESULTS Table I shows the distribution of the various enzymes studied between the kidney cortex and medulla at several stages of development (IO and zo days of age and adult). It can be seen that the enzymes, at all ages studied, can be divided into four groups when classified according to their relative activities in the cortex and medulla. Group I contains the three glycolytic enzymes, all of which show greater activity in the medulla than the cortex. The enzymes from Group 2 are those considered to be gluconeogenie in function and have very high activities in the cortex relative to the medulla. Group 3 consists of the three extramito-
161
chondrial enzymes malate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase, which are present at higher activity in the cortex than the medulla. The final group (Group 4) is made up of ‘ malic enzyme ‘, glucose 6-phosphate dehydrogenase and ATP citrate lyase which are found at equal activities in the two areas. These enzymes are usually considered to be associated with lipogenesis and it was also found (Table I) that acetate incorporation into lipid showed a similar distribution. Detailed developmental patterns have been determined for ‘ malic enzyme ‘, glucose 6-phosphate dehydrogenase and phosphofructokinase. However, since these results merely confirm in more detail the enzyme activity distribution given in Table I and the developmental pattern for whole kidney preparations presented by Hauser & Bailey (1974 a) they have been omitted. DISCUSSION The enzyme distribution studies described in this paper suggest that gluconeogenesis may be almost exclusively located in the kidney cortex. Any gluconeogenic enzyme found in the medulla may be due to contamination because only rough dissections were made and the medullary preparation was more likely to have been contaminated with cortex than vice-versa. The results are in agreement with those of Waldman & Burch (x963) and Szepesi et al. (1970) who found higher activity of gluconeogenic enzymes in the cortex. Therefore most gluconeogenesis occurs in that part of the kidney from which most of the glucose reabsorption occurs, namely the proximal tubules. Indeed, recently, Burch, Lowry, Perry, Fan & Fagioli (1974) have suggested that gluconeogenesis may be entirely restricted to the proximal tubules. Glycogenesis also appears to occur mainly in this area since glycogen synthetase activity is highest in the outer cortex (S&lender, 1973). The fact that malate dehydrogenase exists mainly in the cortex side-by-side with the glucose synthesizing enzymes supports the suggestion that it has a gluconeo-
162
HAUSERAND BAILEY
genie function in kidney, being involved in NADH production. The finding of the three key glycolytic enzymes mainly in the medulla is compatible with the results of Lee, Vance & Cahill (1962) which showed that the metabolism of the medulla in rabbit kidney is almost exclusively glucose-dependent anaerobic glycolysis. Therefore, glycolysis and gluconeogenesis in the kidney appear to be quite well separated spatially and this obviates to a certain extent the need for complicated control systems found in liver cells. However such control does appear to be exercised via pyruvate kinase isoenzymes. Two forms of pyruvate kinase, PKI and PK4, exist in the kidney (Costa, Jimenez De Asua, Rozengurt, Bade & Carminatti, 1972). PK4 is not an allosteric enzyme and is the only form present in the medulla where the metabolism is mainly glycolysis. PK4 is also the major component of the cortical enzyme but there is also some of the allosteric PKr which introduces the possibility of regulating the rate of glycolysis with respect to the rate of gluconeogenesis. The fact that PKI appears during the development of the kidney cortex, at a time when gluconeogenesis is initiated i.e. at birth (Osterman, Fritz & Wuntch, 1973) lends support to such a regulatory role for PKI in this tissue. It is noteworthy that Burch et al. (1974) have shown that the pyruvate kinase of proximal tubules is of low activity and allosterically activated by fructose I ,6-diphosphate. Our results suggest that lipogenesis occurs to the same extent throughout the kidney. Szepesi et al. (1970) reported results similar to those reported here for the distribution of 6-phosphate dehydrogenase and glucose ‘ malic enzyme ’ in the adult kidney. Presumably lipogenesis is controlled in relation to the gluconeogenesis and glycolysis occurring in the cortex and the glycolysis occurring in the medulla.
ACKNOWLEDGEMENTS We thank the Medical Research a research grant to C.A.H.
Council for
Int.
3. Biochem.
REFERENCES BALLARD, F. J., & HANSON, R. W. (Ig67), ‘ Phosphoenolpyruvate and carboxykinase nvruvate carhoxvlase in develoning rat liver ‘, giochem. J., xoq, 866-871. _ BURCH.H. B.. LOWRY. 0. H.. PERRY. S. G.. FAN. L., & FA~IoLI, S. ’ (Ig74); ‘ Effect of a’ge on pyruvate kinase and lactate dehydrogenase distribution in rat kidney ‘, Am. J. Physiol., 226, 1227-1231. COSTA, L., JIMENEZ DE ASUA, L., ROZENGURT, E., BADE, E. G., & CARMINATTI,H. (Ig72), ‘ Allosteric properties of the isoenzymes of pyruvate kinase from rat kidney cortex ‘, Biochim. Biophys. Acta, 289, 128-136. HAUSER,C. A., & BAILEY, E. (x974 a), ‘ Changes in lipogenesis and the activities of some enzymes of glycolysis and lipogenesis in the kidney during development of the rat ‘, ht. J. Biochem., 5, 213-222. HAUSER, C. A., & BAILEY, E. (I974 b), ‘ Changes in the activities of enzvmes of kidnev gluconeogenesis during development of the rat ‘, Int. J. Biochm., In press. KORNBERG,A. (1g55), ‘ Isocitrate Dehydrogenase of Yeast (TPN) ’ in Methodr in Enzymolog (ed. Colowick & Kaplan), Vol. I, p. 705. New York: Academic Press. KREBS, H. A. ( Ig64), ‘ Gluconeogenesis ‘, Proc. R. Sot. B., 159, 545-563. LEE, J. B., VANCE,V. K., & CAHILL,G. F. ( 1962)) ‘ Metabolism of 14C-labelled substrates by rabbit kidney cortex and medulla ‘, Am. .T. Physiol., no3, ;7-36. LOCKWOOD. E. A.. BAILEY. E.. & TAYLOR. C. B. Factors involved in changes in (1974, hepatic lipogenesis during development of the rat ‘, Biochem. J., 1x8, 155-162. OCHOA, S. (Ig55), ‘ Malate Dehydrogenase from Pig Heart ‘, in Methods in Enzymology (ed. Colowick & Kaplan), Vol. I, p. 735. New York: Academic Press. OPIE, L. H., & NEWSHOLME, E. A. (x967), ‘ The I ,6-diphosphatase, fructose activities of phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle ‘, Biochem. J., 103, 391-388. OSTERMAN,J., FRITZ, P. J., & WUNTCH,T. (Ig73), ‘ Pyruvate kinase isozymes from rat tissues. Developmental studies ‘, J. Biol. Chem., 268, 1011-1018. SCHLENDER,K. K. (1g73), ‘ Regulation of renal glycogen synthase interconversion of two forms in vitro ‘, Biochim. Biophys. Acta, 297, 384-398. SMITH, S., & DILS, N. (Ig66), ‘ Fatty acid biosynthesis. Fate of fatty acid synthesized by subcellularfractionsoflactatingrabbitmammary gland ‘, Biochim. Biophys. Acta, 125, 435-44.4. SZEPESI.B.. AVERY, E. H., & FREEDLAND,R. A. (I g7o), ‘-Role of kidney in gluconeogendsis and amino acid catabolism ‘, Am. J. Physiol., 219, 1627-1631.
“
1975,6
KIDNEY ENZYMES DURING DEVELOPMENT
TAYLOR, C. B., BAILEY, E., & BARTLEY, W. (1g67), ‘ Changes in hepatic lipogenesis during development of the rat ‘, Biochem. J., 105,
163
WALKER, D. G., & HOLLAND, G. (Ig65), ‘ The development of hepatic glucokinase in the neonatal rat ‘, Bzi&m. J., 97, 845-854.
717-722. UNDERWOOD, A. H., & NEWSHOLME, E. A. (Ig65), ‘ Properties of phosphofructokinase from rat liver and their relation to the control of glycolysis and gluconeogenesis ‘, Biahem. J., 95, 868-875. WALDMAN, R. H., & BURCH, H. B. (Ig67), ‘ Rapid method for the study of enzyme distribution in rat kidney ‘, Am. 3. Physiol., 204, 749-752.
Key Word Index: Rat, lipogenesis,
glycolysis,
kidney, cortex, gluconeogenesis.
medulla,
THE DISTRIBUTION
OF SOME ENZYMES
GLUCONEOGENESIS DURING
AND LIPOGENESIS DEVELOPMENT
CHRISTINE
A. HAUSER
AND
‘59
OF GLYCOLYSIS, IN THE KIDNEY
OF THE RAT ERNEST
BAILEY
Department of Biochemistry, The University of Sheffield, Sheffield, SIO 2TN, U.K. (Received 19 October 1974) ABSTRACT I. Throughout development the activities of the glycolytic enzymes hexokinase, phosphofructokinase and pyruvate kinase were much greater in the renal medulla than the renal cortex. 2. In contrast, at all ages studied, the activities of the extramitochondrial enzymes aconitate hydratase, malate dehydrogenase and isocitrate dehydrogenase and the gluconeogenic enzymes, fructose x,6-diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase were greater in the cortex than the medulla. 3. Lipogenesis measured by acetate conversion to lipid and the activities of ‘ malic enzyme ‘, glucose 6-phosphate dehydrogenase and ATP citrate lyase were similar in cortex and medulla at various ages. DETAILED developmental patterns for the activities of rat kidney gluconeogenic, glycolytic and lipogenic enzymes have recently been described (Hauser & Bailey, 1974 a, b). However, the enzyme activities were determined in preparations of whole kidneys. Since gluconeogenesis (Krebs, I 964) and some of the enzymes of gluconeogenesis (Waldman & Burch, 1963; Szepesi, Avery & Freedland, 1970) appear to be located in the kidney cortex in the adult animal it was thought necessary to study the distribution of the enzymes of glycolysis, lipogenesis and gluconeogenesis between the kidney cortex and medulla at various stages of development.
MATERIALS
AND METHODS
MATERIALS
The materials used were obtained from the sources described by Hauser & Bailey (I 974 a, b). ANIMALS AND DIET
Details are as described by Hauser & Bailey (1974 a, b) except that only female rats were used. Adult rats weighed about 200 g. MEASUREMENT
OF ENZYME ACTIVITIJIS
The rats were killed by a blow on the head and cervical fracture, between g a.m. and II a.m.
The kidneys were extracted and the capsules removed. The kidney was then dissected into two, transversely, to expose the different regions. Thin slices were cut from the surface of the cortex to give an uncontaminated cortical preparation. A rough dissection was then made along the corticomedullary junction using a fine scalpel and a dissecting magnifying glass. The medulla and papilla appeared to be relatively free of cortical material. Different homogenates of the cortex and medulla samples were prepared for the various enzymes assayed. Homogenization was carried out using a motor-driven glass-Teflon Potter-Elvehjem homogenizer. For the determination of the activity of ‘ malic enzyme ’ (L-malate : NADP oxidoreductase [decarboxylating], EC I. I. I .40), pyruvate kinase (ATP : pyruvate phosphotransferase, EC 2.7. I .40), phosphofructokinase (ATP : n-fructose 6-phosphate I-phosphotransferase, EC 2.7. I. I I), ATP citrate lyase (ATP-citrate oxaloacetate-lyase [CoA acetylating and ATP dephosphorylating], EC 4. I .3.8) and glucose 6-phosphate dehydrogenase (n-glucose 6-phosphate : NADP oxidoreductase, EC I. I. 1.49) tissue homogenates were prepared as described by Lockwood, Bailey & Taylor (x970). For extramitochondrial NADP-dependent isocitrate dehydrogenase (nL-isocitrate : NADP oxidoreductase [decarboxylating], EC I. I. I .42), extramitochondrial malate dehydrogenase (Lmalate : NAD oxidoreductase, EC 1.x.1.37), extramitochondrial aconitate hydratase (citrate [isocitrate] hydrolyase, EC 4.2. I .3), hexokinase (ATP : n-hexose 6_phosphotransferase, EC 2.7. I. I),
8.97 _+0.6 3.48 + 0.4 0.27 If:0.02 3’8+_0.3 2.5 f 0.2 8.9 + 0.5 430f 14 0~15+0~01 0.45 + 0’02 1’0 + 0.04 Ig’o+o’ooI
7.01 + 0.4 1’19 + 0.04 0’ I + 0.03 2.6g+o.I 1*4+0*04
0’ 144 f 0,007 0’2 +_0’01 I’2 + 0.05 16.7 + 0.002
5;2;:;3
1*4+-0.03 1*4+0.1 17’7+ I
20 days
1*3+0*1 1*0+0*2 182+ I
IO days
CORTEX
0*18_+0*02 1.8ko.2 1.6 + 0.02 I 7.2 +_0.002
$4~~‘;’ . . 16.9 z I 5g5+16
8.51_+ 0.4 3’20+0’3 0.4 1 + 0.05 1.24+0*1
1.63fo.1 1.6ko.2 14’752
Adult
0.157 + 0.003 0’2 + 0’02 1.3 + 0.03 ‘3’4+0’01
1’1 kO.03 0.8 f. 0.04 3.6 + 0.2 17554
4’10+-0.4 0.53 + 0.07 0.05 + 0’0 I
3*0+_0.1 1*5*0.1 29.0 f 3
IO days
0~15+0~01 0.40 f 0’02 1.7 _+0.02 17’0 + 0’002
4.76 + 0.07 255 rt IO
1*0+_0.1
I .06 _+0.03
0.07 _+0’0 I
I’oI+o*I
3’27+0’1
2’1+0*1 2’2 + 0’2 2g*6+3
20 days
MEDULLA
0’20 + 0.007 1.8+0*3 I’5 * 0’02 13’gfo.ooI
3’53ZkO.5 0.58 f 0.04 0.09 + 0.008 0.32 + 0.04 1*1g_+0.1 1’1 kO.07 7’2fo.7 343+5
1.5fo.1 3’4kO.3 34’4 f 3
Adult
The results are the means +_SEM of four determinations. Enzyme activities are expressed as pmoles substrate utilized/minute/g wet wt. of tissue. Acetate incorporation is expressed as nmoles [14C]acetate incorporated into lipid/minute/g. wet wt. of tissue.
GLYCOLYSI~ Hexokinase Phosphofructokinase Pyruvate kinase GLUCONEOGENE~I~ Fructose I ,6_diphosphatase Extramitochondrial PEP carboxykinase lviitochondrial PEP carboxykinase Extramitochondrial pyruvate carboxylase Mitochondrial pyruvate carboxylase Extramitochondrial aconitate hydratase Extramitochondrial isocitrate dehydrogenase Extramitochondrial malate dehydrogenase LIPOGENRSIS ATP citrate lyase ‘ Malic enzyme ’ Glucose 6-phosphate dehydrogenase Acetate incorporation into lipid
ENZYME
Table I.-DISTRIBUTION OF ENZYMEAC-~I~ITYIN THE KIDNEY DURINGDEVELOPMENT
Le
$
B
Isem
KIDNEY ENZYMES DURING DEVELOPMENT
acetate incorporation into lipid, tissue homogenates were prepared as described by Hauser & Bailey (1974 a). For fructose 1,6diphosphatase (n-fructose t,6-diphosphate Iphosphohydrolase, EC 3. I .7. I I), phosphoenolpyruvate carboxykinase (GTP : oxaloacetate carboxylase [transphosphorylating], EC 4. I. I .32), and pyruvate carboxylase (pyruvate : carbon dioxide ligase [ADP], EC 6.4. I. I ) , tissue homogenates were prepared as described by Hauser & Bailey (1974 b). In the case of fructose 1,6and
diphosphatase, phosphoenolpyruvate carboxytissue kinase and pyruvate carboxylase, homogenates were fractionated as described by Hauser & Bailey (I 974 b) . For all other enzymes, tissue homogenates were fractionated as described by Hauser & Bailey (1974 a). Fructose r,6-diphosphatase was assayed by the method of Opie & Newsholme (1967). Phosphopyruvate enolpyruvate carboxykinase and carboxylase were assayed by the methods of Ballard & Hanson (1967). Glucose 6-phosphate dehydrogenase, ATP citrate lyase, pyruvate kinase and ‘ malic enzyme ’ were assayed as described by Taylor, Bailey & Bartley (1967). Phosphofructokinase was assayed as described by Underwood & Newsholme (1965). NADPdependent isocitrate dehydrogenase was assayed by the method of Kornberg (1955) and extramitochondrial aconitate hydratase was assayed by the same method except that I mM trisodium citrate was used as substrate instead of DLisocitric acid. Hexokinase was assaved bv the method of Walker & Holland (1965). kxtramitochondrial malate dehydrogenase was assayed as described by Ochoa (1955). All enzyme assays were carried out at 25” C. [U-%]Acetate incorporation into total lipid was measured at 37” C by a modification of the method of Smith & Dils (1966) as described by Hauser & Bailey (I974
a).
RESULTS Table I shows the distribution of the various enzymes studied between the kidney cortex and medulla at several stages of development (IO and zo days of age and adult). It can be seen that the enzymes, at all ages studied, can be divided into four groups when classified according to their relative activities in the cortex and medulla. Group I contains the three glycolytic enzymes, all of which show greater activity in the medulla than the cortex. The enzymes from Group 2 are those considered to be gluconeogenie in function and have very high activities in the cortex relative to the medulla. Group 3 consists of the three extramito-
161
chondrial enzymes malate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase, which are present at higher activity in the cortex than the medulla. The final group (Group 4) is made up of ‘ malic enzyme ‘, glucose 6-phosphate dehydrogenase and ATP citrate lyase which are found at equal activities in the two areas. These enzymes are usually considered to be associated with lipogenesis and it was also found (Table I) that acetate incorporation into lipid showed a similar distribution. Detailed developmental patterns have been determined for ‘ malic enzyme ‘, glucose 6-phosphate dehydrogenase and phosphofructokinase. However, since these results merely confirm in more detail the enzyme activity distribution given in Table I and the developmental pattern for whole kidney preparations presented by Hauser & Bailey (1974 a) they have been omitted. DISCUSSION The enzyme distribution studies described in this paper suggest that gluconeogenesis may be almost exclusively located in the kidney cortex. Any gluconeogenic enzyme found in the medulla may be due to contamination because only rough dissections were made and the medullary preparation was more likely to have been contaminated with cortex than vice-versa. The results are in agreement with those of Waldman & Burch (x963) and Szepesi et al. (1970) who found higher activity of gluconeogenic enzymes in the cortex. Therefore most gluconeogenesis occurs in that part of the kidney from which most of the glucose reabsorption occurs, namely the proximal tubules. Indeed, recently, Burch, Lowry, Perry, Fan & Fagioli (1974) have suggested that gluconeogenesis may be entirely restricted to the proximal tubules. Glycogenesis also appears to occur mainly in this area since glycogen synthetase activity is highest in the outer cortex (S&lender, 1973). The fact that malate dehydrogenase exists mainly in the cortex side-by-side with the glucose synthesizing enzymes supports the suggestion that it has a gluconeo-
162
HAUSERAND BAILEY
genie function in kidney, being involved in NADH production. The finding of the three key glycolytic enzymes mainly in the medulla is compatible with the results of Lee, Vance & Cahill (1962) which showed that the metabolism of the medulla in rabbit kidney is almost exclusively glucose-dependent anaerobic glycolysis. Therefore, glycolysis and gluconeogenesis in the kidney appear to be quite well separated spatially and this obviates to a certain extent the need for complicated control systems found in liver cells. However such control does appear to be exercised via pyruvate kinase isoenzymes. Two forms of pyruvate kinase, PKI and PK4, exist in the kidney (Costa, Jimenez De Asua, Rozengurt, Bade & Carminatti, 1972). PK4 is not an allosteric enzyme and is the only form present in the medulla where the metabolism is mainly glycolysis. PK4 is also the major component of the cortical enzyme but there is also some of the allosteric PKr which introduces the possibility of regulating the rate of glycolysis with respect to the rate of gluconeogenesis. The fact that PKI appears during the development of the kidney cortex, at a time when gluconeogenesis is initiated i.e. at birth (Osterman, Fritz & Wuntch, 1973) lends support to such a regulatory role for PKI in this tissue. It is noteworthy that Burch et al. (1974) have shown that the pyruvate kinase of proximal tubules is of low activity and allosterically activated by fructose I ,6-diphosphate. Our results suggest that lipogenesis occurs to the same extent throughout the kidney. Szepesi et al. (1970) reported results similar to those reported here for the distribution of 6-phosphate dehydrogenase and glucose ‘ malic enzyme ’ in the adult kidney. Presumably lipogenesis is controlled in relation to the gluconeogenesis and glycolysis occurring in the cortex and the glycolysis occurring in the medulla.
ACKNOWLEDGEMENTS We thank the Medical Research a research grant to C.A.H.
Council for
Int.
3. Biochem.
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Key Word Index: Rat, lipogenesis,
glycolysis,
kidney, cortex, gluconeogenesis.
medulla,