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Metabolism of myo-inositol in rabbit kidney and in man
425
BIOCHIMICA ET BIOPHYSICA ACTA BBA
26304
METABOLISM OF MYO-INOSITOL IN RABBIT K I D N E Y AND IN MAN
NORBERT BERNARD
F R E I N K E L , G A B O R A N T O N Y * , H O L L I S R. W I L L I A M S AND R. L A N D A U
Section of Endocrinology and Metabolism, Departments of Medicine and Biochemistry, Northwestern University Medical School, Chicago, Ill., Department of Medicine, Case-Western Reserve University School of Medicine, Cleveland, Ohio and Merck Institute for Therapeutic Research, Rahway, N.J.
(U.S.A.)
(Received O c t o b e r I3th, 1969)
SUMMARY
myo-E2-l~C]Inositol was incubated with slices of rabbit kidney cortex and was administered to a normal subject. Glucose and lactate, in the media from the incubations and in the blood of the subject, were isolated, degraded, and assayed for the 14C distribution in each of their carbons. Distribution of ~4Cin glycerol from the phospholipids in the slices was also determined. ~C was primarily in C-I and C-6 of glucose with greater activity always in C-6 than in C-I. 14C was primarily in C-3 of the lactate and glycerol. These distributions are consistent with gluconeogenesis from myo- II~C]inositol in kidney proceeding via the glucuronic pathway and pentose cycle. This premise is strengthened by a comparison with the labeling patterns in glucose and lactate obtained on incubating kidney slices with E6-~4C]fructose.
INTRODUCTION CHARALAMPOUS et al. 1 found that homogenates of kidney metabolized myoinositol and they isolated an enzyme from kidney which converted myo- I2-14Clinositol to D-~5-14C]glucuronic acid. HOWARD AND ANDERSON2 isolated D-E5-14Clglucuronic acid from kidney slices after incubating them with myo-E2-14Clinositol. Glucose is formed from myo-inositol by rabbit kidney slices (N. FREINKEL, unpublished observation) and this gluconeogenesis can occur from D-glucuronic acid by a well established sequence of reactions involving first the conversion of the D-glucuronic acid to Dxylulose and then subsequent metabolism of this pentose via the pentose cycle 3. POSTERNAK and co-workers4, 5 administered myo-E2-~4Clinositol, myo-E2-3Hl inositol and myo-~6-3Hlinositol to phlorizin-treated rats and found distributions of 14C and '~H in glucose in urine in accord with metabolism by these reactions. P r e s e n t a d d r e s s : D e p a r t m e n t of Medicine a n d Pediatrics, U n i v e r s i t y of V e r m o n t College of Medicine, B u r l i n g t o n , Vt., U.S.A.
Bioehim. Biophys. Acta, 2Ol (197 o) 425-433
N. FREINKEL et al.
426
myo-[2-14C~[nositol metabolized by these reactions would be expected to yield ~6-1~Ciglucose6 : /
3~
*
~ 3 CCCCC*COOH
~ 3 CCCCC*
/
m3o-[Z-14C]inositol D-[5-14C~glucuronic acid D-[5-14C]xylulose
2 CCCCCC* "1 [6-14C]glucose
2 CCCCCC*P [6- lZ;C]fructose-6- P
+ CCC*P [ 3-qd C]glyceraldehyde- 3-P
Isomerization of the E3-14Clglyceraldehyde 3-phosphate to I3-14C;dihydroxyacetone 3-phosphate and then condensation of these triose phosphates, catalyzed by aldolase, should yield Ii,6-14Clglucose as would cleavage of the [6-14C~fructose 6-phosphate to the triose phosphates, their isomerization and recombination. ANDERSON AND COOTS7 gave myo-[2-14Clinositol to rats and 8-12 h post-injection found in each of three experiments about 80% of the ~C in the glucose unit of liver and muscle glycogen in C-I and C-6 of the unit. In two of these degradations, the activity in C-I exceeded that in C-6. While extensive cleavage of [6-14C]fructose 6-phosphate to I3-1~C]triose 3-phosphate and then resynthesis could approach equal labeling in C-I and C-6, there is no good explanation, by presently accepted pathways, for greater activity in C-I than C-6. Indeed, as HOWARD AND ANDERSON2 noted, 14C would have been predicted to be largely in C-6. Thus, glucose from glycogen of white blood cells incubated with D-[5-i%]xylose contains ~4C almost exclusively in C-6 (ref. 8). HOWARD AND ANDERSON2 have attributed the migration into C-I to "secondary processes occurring during the long residence of the glucose in the kidney". In support of this they noted that P. A. WEIN~IOLD (unpublished data) found considerably higher activity in C-6 than C-I of glucose from urine of phlorizin-treated rats given myo-E2-t4C]inositol. Glucose was assumed to remain in the kidney for a longer time in the normal than in the phlorizin-treated rat. However, HOWARD AND ANDERSON2 further noted that there still was substantial activity in C-I and that the mechnism by which this occurred remained to be elucidated. CHAROLLAISAND POSTERNAK¢ found 32.6% of the ~4C in urinary glucose from phlorizin-treated rats given myo[2-~4Clinositol in C-I and C-2 of the glucose and 55.2% in C-6. The distribution patterns in glucose observed by POSTERNAK,ANDERSONAND COOTS and WEINHOLD may be consequent at least in part to metabolism of the labeled myo-inositol in tissues other than kidney and perhaps to some extent from recycling of mvo-inositol resynthesized from glucose 9. We have degraded the glucose and lactate formed slices of rabbit kidney cortex from [2-i*C]myoinositol and compared the distributions with those obtained on incubating E6J~C]fructose with slices and administering myo-[2-~4C]inositol to a normal subject. We have also examined the distribution of label in the glycerol of the phosphoglycerides formed during incubation of kidney slices with myo-[2-14C]-inositol. Biochim. Biophys. Acta, 2 o i (197o) 4 2 5 - 4 3 3
427
I N O S I T O L METABOLISM M A T E R I A L S AND METHODS
Incubation of kidney slices with myo-E2-14C]inositol Slices (o.3-o.4-mm thickness) of the cortices of the kidney from rabbits were incubated in a modified Krebs-Ringer bicarbonate buffer of composition 12o mM NaC1, ~mM KC1, 12 mM MgSO4, 0.8 mM CaC12, 1.2 mM KH2PO~ and 25 mM N a H C Q . The rabbits had either been fed a standard stock diet or fasted 24 h before killing. 450-500 mg of slices were incubated in each flask. The medium contained glucose at a concentration of o, 35 or 12o mg per ioo ml. myo-[2-1aClInositol (purchased from Calbiochem, Lot No. 880435, specific activity 11.8 mC/mmole) was added at a concentration of 8.4 #g/ml. Incubation was I, 3 or 4 h. The slices were then removed and frozen in liquid N2 and the media were then frozen. The media were subsequently thawed and chilled acetone was added in equal volume to aliquots from each medium on ice. The resulting suspensions were centrifuged and 300/~g of each supernatant was applied for chromatography (except for experiments designated 5 and 7 where the media were deproteinized by heat coagulation and 2o0/~g was applied). Chromatography was performed in two dimensions on Whatman No. I filter paper as described elsewhere 1°. The application site was overlaid with 20/~g of glucose. The first dimension was with ionophoresis at 4000 V for 30 rain in pyridine-acetic acid-water (I : IO : 289, by vol.) at pH3.7. The second dimension was with ascending chromatography in isopropanolpyridine-acetic acid-water (8:8: 1:4, by vol.). This clearly resolved lactic acid and the area containing the lactate was cut out and eluted with water. Carrier lactate was added and an aliquot of the solution was evaporated and then combusted to CO2 to give the total 14C activity in the solution. The lactate in the remainder of the solution was degraded 6 to give the activity in each of its three carbons. In the tables, to follow the activity of each of the carbons of lactate is given as a percentage of the sum of the activities in the three carbons which have been set equal to IOO. Percent recovery recorded in the tables is the percentage of the activity in the solution as determined by combustion which this sum represents. Percent recoveries serve as a measure of the adequacy of the isolation and degradation. Recoveries were in general 9O-lOO%. Although the chromatography clearly resolved glucose and myo-inositol, specimens obtained following incubation disclosed some streaking in the glucose area. Accordingly glucose spots were eluted, concentrated and reapplied for ascending chromatography in third dimension in n-butanol-ethanol-water (52:3o:18, by vol.). The glucose spots were then cut out and eluted with water, an aliquot combusted to CO2 and the 14C activity in the CO2 determined. The remainder of the solution was then incubated with Leuconostoc mesenteroides to yield C-I of glucose as CO2, C-2 and C-3 as ethanol and C-4, C-5 and C-6 as lactate *. The ethanol and lactate were degraded as previously described to yield each of the carbons of glucose as CO~ and the C02 was assayed for 14C activity. Results recorded in the tables to follow are as for lactate except that the sum of the activities in the six carbons has been set equal to IOO. Percent recoveries are the percent of 14C in the solution recovered in the individual carbons of glucose. In general, recoveries were poor and because of this, for each of two samples, a glucosazone was made from an aliquot. The activity in the glucosazone was in good agreement with the sum of the activities in the individual Biochim. Biophys. Acta, 2Ol (197 o) 4 2 5 - 4 3 3
428 TABLE
N. F R E I N K E L
et
al.
I
DISTRIBUTION OF 14C IN GLUCOSE FORMED FROM myo-[2-14C]INOSITOL BY RABBIT KIDNEY SLICES
Expt. Conditions 14C in glucose carbon No. Dietary Medium Incubation C-r C-2 C-3 C-4 state glucose period (r~g/±oo ml) (h) I 2 3 4** 5 6
Fed Fed Fed Fed Fast Fast
35 12o 35 12o 35 35
I I 4 3 i 4
4o.9 3.1 31.3 7.8 17.6 ii.I 25-5 +. . . . 24.8 7.4 16.5 7.7
1. 7 2.o 6.0
C-5
2. 3 1.8 3.2 3.2 4.8 lO. 3 11-4 - - - - - - - + 2.3 2.9 3-9 3.6 5-5 5.8
C-6
5o.2 52.5 50.2 Lost 58,7 60. 9
Recovery (o)
8o. 4 (94.1)* 82.1 52.0 7 3 . 4 (lOO.2) * 82.1
* Recovery in osazone in parentheses. * * A c t i v i t i e s o f C - I a n d C - 2 - C - 5 a r e r e c o r d e d a s t h e p e r c e n t of t h e a c t i v i t y in t h e g l u c o s e o b t a i n e d on its combustion.
carbons. This is evidence that the activities attributed to the individual carbons of glucose were indeed of glucose and that a non-osazone forming radioactive compound, contaminating the sample, did not yield 1'C in the products of degradation. MU~TZ11 has also noted the specificity of the degradation in the presence of contaminating substances. In one experiment (Table I, Expt. 4) the glucose was converted to gluconic acid with glucose oxidase and the gluconic acid purified and degraded with H I Q to yield C-I as C02, C-2-C-5 as formic acid and C-6 as formaldehyde and these products were assayed for 1~C (ref. 12). The evidence for the adequacy of the procedure was established by degradations of [I-14C]glucose and [6-1~C]glucose. Slices were homogenized in a glass homogenizer containing 4 ml of chloroformmethanol (2 : I, by vol.). The homogenates were centrifuged, the volume adjusted to exactly io ml with chloroform-methanol (2:1, by vol.), and the phases separated with 2 ml of water 1~. The lipid extract was washed three additional times with "upper phase" (ref. 1 3 ) and partitioned into neutral lipids and phospholipids as previously described 1.. The phospholipids were subiected to mild alkaline hydrolysis and the water-soluble glycerylphosphoryl-base units isolated 15. Glycerylphosphorylcholine and glycerylphosphorylethanolamine were eluted, pooled and then hydrolyzed for 24 h with 5 ml 2 M HC1 after adding 2o/~g of carrier glycerol. The hydrolysates were taken to dryness in vacuo to remove HC1. They were resuspended in water and then chromatographed two-dimensionally on Whatman No. I filter paper with phenolwater-acetic acid-ethanol (9: I : I : 1.2, by vol.) descending followed by isopropanolpyridine-acetic acid-water (I :8:1:4, by vol.) ascending. The spots were localized by radioautography and only two radioactive spots were discernible. One spot had the chromatographic and staining characteristics of glycerol reference markers and it was cut out, and designated as "glycerol". The second had the characteristics of glycerophosphate. To document its identity further, it was eluted and reapplied for ionophoresis at 4ooo V for 3o min in pyridine-acetic acid-water (I : io : 289, by vol.) at pH 3-7. It remained homogeneous and displayed the ionophoretic characteristics of glycerophosphate. It was therefore cut out and designated "glycerophosphate". Carrier glycerol was added to an aqueous extract of the glycerol spots and the resulting solutions were incubated with Aerobacter aerogenes. This bacteria converts glycerol to lactate and the lactate was isolated and degraded and the resulting CQ's Biochim. Biophys. Acta, 2Ol (197 o) 4 2 5 - 4 3 3
429
INOSITOL METABOLISM
assayed for ~4C (ref. 6). One glycerophosphate sample was hydrolyzed by incubation with alkaline phosphatase. The hydrolyzate, after addition of carrier glycerol, was passed down a mixed-bed ion-exchange resin (Amberlite MB-3, Rohm and Haas Co., Philadelphia, Pa., U.S.A.) and the glycerol in the effluent was degraded using A.
aerogenes. Incubations qf kidney slices with o-[6-~C]fructose In one experiment D-[6-~4C]fructose (117.6/~g/ml, 0.8 #C/ml purchased from Nuclear Research Corp., Orlando, Fla.) was the labeled substrate rather than myo[2-14C]inositol, but unlabeled myo-inositol was added at 8.4 /~g/ml. Glucose was present at a concentration of 35 mg per IOO ml. Two flasks were incubated, one with slices of cortex from a fed rabbit, the other with slices from a fasted rabbit and the former incubation was for I h and the latter for 4 h. At the completion of incubation the media were heat coagulated and filtered. Analysis by a glucose oxidase method showed 9.8 m g / I o o ml glucose remaining in the flask incubated for i h and 0.8 mg per IOO ml in that for 4 h. A portion of the medium was chromatographed in two dimensions as described above. The lactate spot was then eluted and the lactate degraded and assayed. The remainder of the media was passed through a column packed with Amberlite MB- 3. Effluent and aqueous washings from the column were evaporated to a small volume which was applied to W h a t m a n 3 MM paper. Descending chromatography was performed using a n-butanol-acetic acid-water (4: i :5, by vol.) system with fructose and glucose guide spots. The area corresponding to glucose was eluted and the eluate was passed through another Amberlite MB-3 column. Carrier glucose was added and the solution degraded using Leuconostoc mesenteroides. A radioactive non-osazone forming contaminant was again present. This was considered to possibly be sorbitol, but treatment of an aliquot of the solution with sorbitol dehydrogenase did not yield radioactive fructose on chromatography.
Administration of myo-[2-'4C]inositol to a normal volunteer myo-[2-14C]Inositol (Calbiochem,. Lot No. C-Io36i, specific activity I mC/mM), 50 /~C, after autoclaving in saline was injected intravenously into a 23-year-old female volunteer (wt. 47.2 kg, height 5'3") who had been fasted overnight. IOO ml of blood was drawn at I , 2 and 3 h after the injection. The blood was introduced into 700 ml of water and precipitated with IOO ml o.15 M Ba(OH)~ and ioo ml 5~o ZnSO4 (ref. 16). The supernatants from the samples obtained at I and 3 h were passed through a column of Duolite-C-3 in the H + form and Duolite A- 4 in the O H - form (Diamond Alkali Co., Redwood City, Calif., U.S.A.). The effluent and aqueous washings of the column were evaporated to a small volume which was applied to W h a t m a n 3MM paper and chromatographed in the butanol acetic acid-water system. The glucose was isolated, degraded and the degradation products assayed for 14C as described above. The supernatant fluid from the 2-h period was evaporated to about 20 ml, acidified to p H I with o.5 M H2S04, carrier lactate was added and the solution was then continuously extracted with diethyl ether. The diethyl ether extract was neutralized with I M NaOH and evaporated. The lactate in the residue was purified on a celite column ~7 and then degraded and assayed as described above. Biochim. Biophys. Acta, 2Ol (197 o) 425-433
N. FREINKEL e{ al.
430 RESULTS
The distribution of 14C on the glucose formed from rnyo-[12-14C]inositol (Table I) by the slices of kidney showed activity primarily in C-I and C-6. There was 4o-8o% as much activity in C-I as in C-6 in the I-h experiments and 25-35% as much activity in the 4-h experiments. There was more activity in C-2-C- 5 in the experiments of 4-h duration. No differences as a function of dietary state or the concentration of glucose in the medium are apparent. The 14C in the glucose from the blood of the normal subject given m3,o-~2J~C ~inositol (Table II) also was primary in C-I and C-6. There was 70 and 85% as much activity in C-I as in C-6 in the glucose from the blood drawn I and 3 11, respectively, after the myo-ii2-14C]inositol injection and greater randomization of nC into C-2 C-5 at 3 h than I h. Lactate isolated from the medium after the incubations of Expts. I and 5 (Table I I I ) contained 14C primarily in C-3 while the lactate from the blood of the normal subject (drawn 2 h after the mvo-!2-14Clinositol administration) has 70% as much activity in C-I as C-3. Glycerol isolated from the phospholipid fraction contained activity primarily in C-3 (Table IV). T A B L E 1I DISTRIBUTION
OF
14C I N
BLOOD
GLUCOSE
FROM
NORMAL
SUBJECT
ADMINISTERED
tllyo-I2-14C I-
INOSITOL
Time after injection (h)
14C in glucose carbon C-Jr C-2 C-3
C-4
C-5
C-6
Recovery (%)
I
35,o 35 .8
2.6 4 .8
5.0 7 .8
51.9 41'8
94.8 l°°'3
3
4.0 5 .2
1. 5 4 .6
TABLE III DISTRIBUTION
O F 14C I N L A C T A T E F O R M E D F R O M m y 0 - [ 2 - 1 4 C ] I N O S I T O L
BY KIDNEY
SLICES AND THE
NORMAL SUBJECT
Source
1~C in lactate carbon
Expt. I Expt. 5 Normal subject
Recovery
c-f
c-2
c-3
(%)
6.0 5.4 36. i
6.6 6.1 12. i
87. 4 88.5 51.8
87.6 98.o i oo.2
T A B L E IV DISTRIBUTION
O F 14C I N G L Y C E R O L F O R M E D F R O M
myo-E2-14CilNOSITOL
Expt. No.
14C in glycerol carbon C-r C-2 C-3
Recovery (°/o)
2 5 6 7*
34.4 5.6 15.3 31-7
lO6.9 112.o** 92.6 96"5
6.3 9.9 9.3 7 .0
59.3 84.5 75.4 61"3
BY RABBIT KIDNEY
SLICES
* I n E x p t . 7 only glycerol w a s d e g r a d e d . Slices were from fed r a b b i t s , i n c u b a t i o n w a s for 3 h a n d was done in a m e d i u m n o t p r e p a r e d w i t h glucose. ** Glycerol was i s o l a t e d from g l y c e r o p h o s p h a t e b y t r e a t m e n t w i t h a l k a l i n e p h 0 s p h a t a s e .
Biochim. Biophys. Acta, 2Ol (197 o) 425-433
431
I N O S I T O L METABOLISM TABLE
V
DISTRIBUTION IN GLUCOSE AND LACTATE FORMED FROM D-[6-14CJFRUCTOSE BY RABBIT KIDNEY SLICES
Dietary state
Period of incubation (o//)
14C in glucose carbon C-t C-2 C-3 C-4 C-5 C-6
Recovery (%)
1~C in lactate carbon C-± C-2 C-3
Recovery (%)
Fed Fasted
I 4
19.3 17.2
66.2 ( l O l . 4 ) * 16.7 (8o-4)*
o.o lO.4
lOl.9 98. 5
4.6 6.6
o.7 3.0
2.1 2.9
i.t 3.3
72.2 67.0
4.8 9-4
95.2 80.2
* R e c o v e r e d i n o s a z o n e in p a r e n t h e s e s .
In the incubations of [6-14C]fructose with rabbit slices (Table V) there was onefourth as much 14C in C-I as C-6 and again 14C was primarily in these carbons. Incorporation of 14C in lactate was primarily in C-3. DISCUSSION
In no instance have we observed a greater activity in C-I than C-6 of glucose as was reported by ANDERSON AND COOTS7. The distributions in glucose we observe are in accord with the metabolism via the glucuronic acid pathway and pentose cycle. These results document in vitro what has been presumed to occur in the kidney from studies in vivo. In the slice experiment (Table I) the incorporation into C-I can be attributed to both the equilibration of the E3-14Clglyceraldehyde 3-phosphate formed via these pathways (see INTRODUCTION) with dihydroxyacetone 3-phosphate and then formation of glucose, and the cleavage of fructose 6-phosphate formed from the myo-E2-14C~inositol into the triose phosphates, their equilibration and then formation of glucose, i.e. : [6- 14C]fructose-6-P ----~[3- 14C]glyceraldehyde-3-P dihydroxyacetone-3-P
[1,6- 14(2 ]fructose-6-P [1,6- 14C ]glucose
The significance of the lesser incorporation into C-I relative to C-6 after 4 h of incubation than after the earlier time periods is uncertain because of the limited number of experiments performed. The extent of incorporation into C-I compared to C-6 would be dependent upon the rate of isomerization of glyceraldehyde 3-phosphate to dihydroxyacetone 3-phosphate relative to the rate of formation of the glucose and these rates could have changed with time. The greater activity in C-2-C-5 at 4 h than at the earlier hours is in accord with increasing randomization with time of the 14C into oxaloacetate and pyruvate via the reactions of the Krebs cycle and the synthesis of glucose from these compounds. The distribution of 1~C in lactate (Table II) is also in accord with such metabolism with the ~4C in C-3 reflecting formation from E3-14C]glyceraldehyde 3-phosphate and the 14C in C-I and C-2 reflecting randomization via the reactions of the Krebs cycle. Since myo-inositol can be synthesized from glucose by kidney slices ~8, the 14C distributions could conceivably be consequent in some degree to the metabolism of 14C-labeled myo-inositol formed from the 14C-labeled glucose in turn formed from the myo-~2-1~CJinositol added as labeled substrate. This contribution was almost certainly negligible in consideration of the dilution of the ~4C of the myo-[2-1*C~inositol during this recycling and the limited amount of conversion of glucose to mD,o-inositoltS. The distributions of I4C in the glucose from the blood of the subject given n(voBiochim. Biophys. Acta, 2Ol (197 o) 4 2 5 - 4 3 3
432
N. FREINKEL et al.
E2-i4C]inositol are similar to those obtained using the kidney slices, though there perhaps was a tendency to greater incorporation into C-I relative to C-6 by the subject. Most probably the glucose in the blood was formed primarily from myo-i2-I4C] inositol without lactate as an intermediate. If the glucose has been formed from [3-14Cllactate one would expect glucose with 14C extensively in C-2-C-5 in accord with the metabolism of the carbons of lactate in the Krebs cycle prior to their conversion to glucose I9. C-I to C-6 ratios when [6-I4C]Iructose was substrate were in general lower than when myo-E2-i4Clinositol was substrate. If in kidney the [6-i4C]fructose was phosphorylated to [6-14C]fructose 6-phosphate rather than [6-14Clfructose 1-phosphate this would be expected since incorporation into C-I from the [6-i4Clfructose could then only occur through cleavage to the triose phosphates and then resynthesis. The data would indicate that much of [6-14Clfructose was phosphorylated to [6-I4Cifructose 6-phosphate since each molecule of [6-14C]fructose 1-phosphate would yield in its further metabolism a molecule of I3-1~C~glyceraldehyde 3-phosphate and this would then favor increased incorporation into C-I. In accord with this the activity ill the rat of the enzyme fructokinase, catalyzing the formation of fructose 1-phosphate, is several-fold lower in kidney than in liver ~°. In liver where the fructose I-phosphate pathway is so active, C-I of glucose formed from E6-i~Clfructose does have 7o-8o% of the activity of C-6 (ref. ii). This is also the case for blood glucose when i6-1~C!fructose is administered to man (J. MARSHALL, J. CRAIG, K. HOSTETLERAND B. R. LANDAU, unpublished observations). The considerable activity in C-I in two degradations of glycerol formed by kidney slices and in the blood lactate from the subject given myo-~2-I4Clinositol remain unexplained. The glycerols would be expected to reflect the distribution in glyceraldehyde 3-phosphate and hence have activity primarily in C-3 and the lactate should be expected to have a distribution similar to that seen in the experiments in vitro. Note added in proof (Received D e c e m b e r i8th, 1969) Since submission of this p a p e r H a n k e s et al. ~1 have reported a s t u d y of the m e t a b o l i s m of myoinositol in h u m a n s . I n three n o r m a l subjects three nonpentosuric kindred of subjects w i t h pentosuria, two h o u r s after a d m i n i s t r a t i o n of myo-F2-a4C] inositol 3I :~ 2 % and 48 Jz 3% of the 14C in blood glucose was recovered in C-I and C-6 respectively, a distribution similar to t h a t in o u r one subject. ACKNOWLEDGMENTS
This study was supported by Research Grants AM-Io627 and AM-Io699 and by Training Grant AM-5o7I from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service, Bethesda, Md., U.S.A. REFERENCES I E. C. CHARALAMPOUS, S, 13UMILLER AND S. GRAHAM, J. Am. Chem. Soc., 80 (1958) 2022. 2 C. F. HOWARD, Jr. AND L. ANDERSON, Arch. Biochem. Biophys. 118 (1967) 332. 3 J. J- BURNS, in D. M. GREENBERG, Metabolic Pathways, Academic Press, New York, 3rd Ed., 1967, p. 405. 4 E. CHAROLLAIS AND T. POSTERNAK, Helv. Chim. Acta, 48 (1965) 280. 5 T. POSTERNAK, The Cyclitols, Holden-Day, San Francisco, 1965, p. 329 . 6 ]3. R. LANDAU, C*. E. BARTSCH AND H. R. WILLIAMS, J. Biol. Chem., 241 (1966) 75 o. 7 L. ANDERSON AND R. H. COOTS, Biochim. Biophys. Acta, 28 (1958) 666.
Biochim. Biophys. Acta, 2Ol (i97o) 425-433
INOSITOL METABOLISM
433
8 R. STJERNHOLM AND E. P. NOBLE, J. Biol. Chem., 236 (1966) 6I 4, 9 N. FREINKEL AND R. M. C. DAWSON, Biochem. J., 81 (1961) 250. IO N. FREINKEL, A. I~. COHEN, R. A. ARKY AND A. E. FOSTER, J. Clin. Endocrinol. Metab., 25 (1965) 76 . i i J. A. MUNTZ, J. Biol. Chem., 243 (1968) 2788. 12 L. W. WHITE AND t3. R. LANDAU, J. Clin. Invest., 44 (1965) 12oo. 13 J. FOLCH, M. LEES AND G. H. SLOANE-STANLEY, J. Biol. Chem., 226 (1957) 497. 14 N. FREINKEL, A. D. LITONJUA AND E. C. SAEF, Nature, 191 (1961) 804. 15 R. YI. C. DAWSON, N. HEMINGTON AND J. B. DAVENPORT, Biochem. J., 84 (1962) 49716 M. SOMOGYI, J. Biol. Chem., 195 (1952) 19. 17 E. H. SWIM AI','p L. O. KRAMPITZ, J. Bacteriol., 67 (1954) 419 • 18 G. ]:-IAUSER AND V. N. FINELLI, J. Biol. Chem., 238 (1963) 3224 . 19 K. Y. HOSTETLER, H. R. WILLIAMS, W. W. SHREEVE AND B. R. LANDAU, J. Biol. Chem., 244 (1969) 2075. 20 R. C. ADELMAN, F. J. BALLARD AND S. V~rEINHOUSE, J. Biol. Chem., 242 (1967) 3360. 21 L. V. HANKES, W. M. POLIBZER, O. TOUSTER, AND L. ANDERSON, Ann. N . Y , Acad. Sci., 165 (I969) 564 .
Biochim. Biophys. Acta, 2Ol (197 o) 425-433
BIOCHIMICA ET BIOPHYSICA ACTA BBA
26304
METABOLISM OF MYO-INOSITOL IN RABBIT K I D N E Y AND IN MAN
NORBERT BERNARD
F R E I N K E L , G A B O R A N T O N Y * , H O L L I S R. W I L L I A M S AND R. L A N D A U
Section of Endocrinology and Metabolism, Departments of Medicine and Biochemistry, Northwestern University Medical School, Chicago, Ill., Department of Medicine, Case-Western Reserve University School of Medicine, Cleveland, Ohio and Merck Institute for Therapeutic Research, Rahway, N.J.
(U.S.A.)
(Received O c t o b e r I3th, 1969)
SUMMARY
myo-E2-l~C]Inositol was incubated with slices of rabbit kidney cortex and was administered to a normal subject. Glucose and lactate, in the media from the incubations and in the blood of the subject, were isolated, degraded, and assayed for the 14C distribution in each of their carbons. Distribution of ~4Cin glycerol from the phospholipids in the slices was also determined. ~C was primarily in C-I and C-6 of glucose with greater activity always in C-6 than in C-I. 14C was primarily in C-3 of the lactate and glycerol. These distributions are consistent with gluconeogenesis from myo- II~C]inositol in kidney proceeding via the glucuronic pathway and pentose cycle. This premise is strengthened by a comparison with the labeling patterns in glucose and lactate obtained on incubating kidney slices with E6-~4C]fructose.
INTRODUCTION CHARALAMPOUS et al. 1 found that homogenates of kidney metabolized myoinositol and they isolated an enzyme from kidney which converted myo- I2-14Clinositol to D-~5-14C]glucuronic acid. HOWARD AND ANDERSON2 isolated D-E5-14Clglucuronic acid from kidney slices after incubating them with myo-E2-14Clinositol. Glucose is formed from myo-inositol by rabbit kidney slices (N. FREINKEL, unpublished observation) and this gluconeogenesis can occur from D-glucuronic acid by a well established sequence of reactions involving first the conversion of the D-glucuronic acid to Dxylulose and then subsequent metabolism of this pentose via the pentose cycle 3. POSTERNAK and co-workers4, 5 administered myo-E2-~4Clinositol, myo-E2-3Hl inositol and myo-~6-3Hlinositol to phlorizin-treated rats and found distributions of 14C and '~H in glucose in urine in accord with metabolism by these reactions. P r e s e n t a d d r e s s : D e p a r t m e n t of Medicine a n d Pediatrics, U n i v e r s i t y of V e r m o n t College of Medicine, B u r l i n g t o n , Vt., U.S.A.
Bioehim. Biophys. Acta, 2Ol (197 o) 425-433
N. FREINKEL et al.
426
myo-[2-14C~[nositol metabolized by these reactions would be expected to yield ~6-1~Ciglucose6 : /
3~
*
~ 3 CCCCC*COOH
~ 3 CCCCC*
/
m3o-[Z-14C]inositol D-[5-14C~glucuronic acid D-[5-14C]xylulose
2 CCCCCC* "1 [6-14C]glucose
2 CCCCCC*P [6- lZ;C]fructose-6- P
+ CCC*P [ 3-qd C]glyceraldehyde- 3-P
Isomerization of the E3-14Clglyceraldehyde 3-phosphate to I3-14C;dihydroxyacetone 3-phosphate and then condensation of these triose phosphates, catalyzed by aldolase, should yield Ii,6-14Clglucose as would cleavage of the [6-14C~fructose 6-phosphate to the triose phosphates, their isomerization and recombination. ANDERSON AND COOTS7 gave myo-[2-14Clinositol to rats and 8-12 h post-injection found in each of three experiments about 80% of the ~C in the glucose unit of liver and muscle glycogen in C-I and C-6 of the unit. In two of these degradations, the activity in C-I exceeded that in C-6. While extensive cleavage of [6-14C]fructose 6-phosphate to I3-1~C]triose 3-phosphate and then resynthesis could approach equal labeling in C-I and C-6, there is no good explanation, by presently accepted pathways, for greater activity in C-I than C-6. Indeed, as HOWARD AND ANDERSON2 noted, 14C would have been predicted to be largely in C-6. Thus, glucose from glycogen of white blood cells incubated with D-[5-i%]xylose contains ~4C almost exclusively in C-6 (ref. 8). HOWARD AND ANDERSON2 have attributed the migration into C-I to "secondary processes occurring during the long residence of the glucose in the kidney". In support of this they noted that P. A. WEIN~IOLD (unpublished data) found considerably higher activity in C-6 than C-I of glucose from urine of phlorizin-treated rats given myo-E2-t4C]inositol. Glucose was assumed to remain in the kidney for a longer time in the normal than in the phlorizin-treated rat. However, HOWARD AND ANDERSON2 further noted that there still was substantial activity in C-I and that the mechnism by which this occurred remained to be elucidated. CHAROLLAISAND POSTERNAK¢ found 32.6% of the ~4C in urinary glucose from phlorizin-treated rats given myo[2-~4Clinositol in C-I and C-2 of the glucose and 55.2% in C-6. The distribution patterns in glucose observed by POSTERNAK,ANDERSONAND COOTS and WEINHOLD may be consequent at least in part to metabolism of the labeled myo-inositol in tissues other than kidney and perhaps to some extent from recycling of mvo-inositol resynthesized from glucose 9. We have degraded the glucose and lactate formed slices of rabbit kidney cortex from [2-i*C]myoinositol and compared the distributions with those obtained on incubating E6J~C]fructose with slices and administering myo-[2-~4C]inositol to a normal subject. We have also examined the distribution of label in the glycerol of the phosphoglycerides formed during incubation of kidney slices with myo-[2-14C]-inositol. Biochim. Biophys. Acta, 2 o i (197o) 4 2 5 - 4 3 3
427
I N O S I T O L METABOLISM M A T E R I A L S AND METHODS
Incubation of kidney slices with myo-E2-14C]inositol Slices (o.3-o.4-mm thickness) of the cortices of the kidney from rabbits were incubated in a modified Krebs-Ringer bicarbonate buffer of composition 12o mM NaC1, ~mM KC1, 12 mM MgSO4, 0.8 mM CaC12, 1.2 mM KH2PO~ and 25 mM N a H C Q . The rabbits had either been fed a standard stock diet or fasted 24 h before killing. 450-500 mg of slices were incubated in each flask. The medium contained glucose at a concentration of o, 35 or 12o mg per ioo ml. myo-[2-1aClInositol (purchased from Calbiochem, Lot No. 880435, specific activity 11.8 mC/mmole) was added at a concentration of 8.4 #g/ml. Incubation was I, 3 or 4 h. The slices were then removed and frozen in liquid N2 and the media were then frozen. The media were subsequently thawed and chilled acetone was added in equal volume to aliquots from each medium on ice. The resulting suspensions were centrifuged and 300/~g of each supernatant was applied for chromatography (except for experiments designated 5 and 7 where the media were deproteinized by heat coagulation and 2o0/~g was applied). Chromatography was performed in two dimensions on Whatman No. I filter paper as described elsewhere 1°. The application site was overlaid with 20/~g of glucose. The first dimension was with ionophoresis at 4000 V for 30 rain in pyridine-acetic acid-water (I : IO : 289, by vol.) at pH3.7. The second dimension was with ascending chromatography in isopropanolpyridine-acetic acid-water (8:8: 1:4, by vol.). This clearly resolved lactic acid and the area containing the lactate was cut out and eluted with water. Carrier lactate was added and an aliquot of the solution was evaporated and then combusted to CO2 to give the total 14C activity in the solution. The lactate in the remainder of the solution was degraded 6 to give the activity in each of its three carbons. In the tables, to follow the activity of each of the carbons of lactate is given as a percentage of the sum of the activities in the three carbons which have been set equal to IOO. Percent recovery recorded in the tables is the percentage of the activity in the solution as determined by combustion which this sum represents. Percent recoveries serve as a measure of the adequacy of the isolation and degradation. Recoveries were in general 9O-lOO%. Although the chromatography clearly resolved glucose and myo-inositol, specimens obtained following incubation disclosed some streaking in the glucose area. Accordingly glucose spots were eluted, concentrated and reapplied for ascending chromatography in third dimension in n-butanol-ethanol-water (52:3o:18, by vol.). The glucose spots were then cut out and eluted with water, an aliquot combusted to CO2 and the 14C activity in the CO2 determined. The remainder of the solution was then incubated with Leuconostoc mesenteroides to yield C-I of glucose as CO2, C-2 and C-3 as ethanol and C-4, C-5 and C-6 as lactate *. The ethanol and lactate were degraded as previously described to yield each of the carbons of glucose as CO~ and the C02 was assayed for 14C activity. Results recorded in the tables to follow are as for lactate except that the sum of the activities in the six carbons has been set equal to IOO. Percent recoveries are the percent of 14C in the solution recovered in the individual carbons of glucose. In general, recoveries were poor and because of this, for each of two samples, a glucosazone was made from an aliquot. The activity in the glucosazone was in good agreement with the sum of the activities in the individual Biochim. Biophys. Acta, 2Ol (197 o) 4 2 5 - 4 3 3
428 TABLE
N. F R E I N K E L
et
al.
I
DISTRIBUTION OF 14C IN GLUCOSE FORMED FROM myo-[2-14C]INOSITOL BY RABBIT KIDNEY SLICES
Expt. Conditions 14C in glucose carbon No. Dietary Medium Incubation C-r C-2 C-3 C-4 state glucose period (r~g/±oo ml) (h) I 2 3 4** 5 6
Fed Fed Fed Fed Fast Fast
35 12o 35 12o 35 35
I I 4 3 i 4
4o.9 3.1 31.3 7.8 17.6 ii.I 25-5 +. . . . 24.8 7.4 16.5 7.7
1. 7 2.o 6.0
C-5
2. 3 1.8 3.2 3.2 4.8 lO. 3 11-4 - - - - - - - + 2.3 2.9 3-9 3.6 5-5 5.8
C-6
5o.2 52.5 50.2 Lost 58,7 60. 9
Recovery (o)
8o. 4 (94.1)* 82.1 52.0 7 3 . 4 (lOO.2) * 82.1
* Recovery in osazone in parentheses. * * A c t i v i t i e s o f C - I a n d C - 2 - C - 5 a r e r e c o r d e d a s t h e p e r c e n t of t h e a c t i v i t y in t h e g l u c o s e o b t a i n e d on its combustion.
carbons. This is evidence that the activities attributed to the individual carbons of glucose were indeed of glucose and that a non-osazone forming radioactive compound, contaminating the sample, did not yield 1'C in the products of degradation. MU~TZ11 has also noted the specificity of the degradation in the presence of contaminating substances. In one experiment (Table I, Expt. 4) the glucose was converted to gluconic acid with glucose oxidase and the gluconic acid purified and degraded with H I Q to yield C-I as C02, C-2-C-5 as formic acid and C-6 as formaldehyde and these products were assayed for 1~C (ref. 12). The evidence for the adequacy of the procedure was established by degradations of [I-14C]glucose and [6-1~C]glucose. Slices were homogenized in a glass homogenizer containing 4 ml of chloroformmethanol (2 : I, by vol.). The homogenates were centrifuged, the volume adjusted to exactly io ml with chloroform-methanol (2:1, by vol.), and the phases separated with 2 ml of water 1~. The lipid extract was washed three additional times with "upper phase" (ref. 1 3 ) and partitioned into neutral lipids and phospholipids as previously described 1.. The phospholipids were subiected to mild alkaline hydrolysis and the water-soluble glycerylphosphoryl-base units isolated 15. Glycerylphosphorylcholine and glycerylphosphorylethanolamine were eluted, pooled and then hydrolyzed for 24 h with 5 ml 2 M HC1 after adding 2o/~g of carrier glycerol. The hydrolysates were taken to dryness in vacuo to remove HC1. They were resuspended in water and then chromatographed two-dimensionally on Whatman No. I filter paper with phenolwater-acetic acid-ethanol (9: I : I : 1.2, by vol.) descending followed by isopropanolpyridine-acetic acid-water (I :8:1:4, by vol.) ascending. The spots were localized by radioautography and only two radioactive spots were discernible. One spot had the chromatographic and staining characteristics of glycerol reference markers and it was cut out, and designated as "glycerol". The second had the characteristics of glycerophosphate. To document its identity further, it was eluted and reapplied for ionophoresis at 4ooo V for 3o min in pyridine-acetic acid-water (I : io : 289, by vol.) at pH 3-7. It remained homogeneous and displayed the ionophoretic characteristics of glycerophosphate. It was therefore cut out and designated "glycerophosphate". Carrier glycerol was added to an aqueous extract of the glycerol spots and the resulting solutions were incubated with Aerobacter aerogenes. This bacteria converts glycerol to lactate and the lactate was isolated and degraded and the resulting CQ's Biochim. Biophys. Acta, 2Ol (197 o) 4 2 5 - 4 3 3
429
INOSITOL METABOLISM
assayed for ~4C (ref. 6). One glycerophosphate sample was hydrolyzed by incubation with alkaline phosphatase. The hydrolyzate, after addition of carrier glycerol, was passed down a mixed-bed ion-exchange resin (Amberlite MB-3, Rohm and Haas Co., Philadelphia, Pa., U.S.A.) and the glycerol in the effluent was degraded using A.
aerogenes. Incubations qf kidney slices with o-[6-~C]fructose In one experiment D-[6-~4C]fructose (117.6/~g/ml, 0.8 #C/ml purchased from Nuclear Research Corp., Orlando, Fla.) was the labeled substrate rather than myo[2-14C]inositol, but unlabeled myo-inositol was added at 8.4 /~g/ml. Glucose was present at a concentration of 35 mg per IOO ml. Two flasks were incubated, one with slices of cortex from a fed rabbit, the other with slices from a fasted rabbit and the former incubation was for I h and the latter for 4 h. At the completion of incubation the media were heat coagulated and filtered. Analysis by a glucose oxidase method showed 9.8 m g / I o o ml glucose remaining in the flask incubated for i h and 0.8 mg per IOO ml in that for 4 h. A portion of the medium was chromatographed in two dimensions as described above. The lactate spot was then eluted and the lactate degraded and assayed. The remainder of the media was passed through a column packed with Amberlite MB- 3. Effluent and aqueous washings from the column were evaporated to a small volume which was applied to W h a t m a n 3 MM paper. Descending chromatography was performed using a n-butanol-acetic acid-water (4: i :5, by vol.) system with fructose and glucose guide spots. The area corresponding to glucose was eluted and the eluate was passed through another Amberlite MB-3 column. Carrier glucose was added and the solution degraded using Leuconostoc mesenteroides. A radioactive non-osazone forming contaminant was again present. This was considered to possibly be sorbitol, but treatment of an aliquot of the solution with sorbitol dehydrogenase did not yield radioactive fructose on chromatography.
Administration of myo-[2-'4C]inositol to a normal volunteer myo-[2-14C]Inositol (Calbiochem,. Lot No. C-Io36i, specific activity I mC/mM), 50 /~C, after autoclaving in saline was injected intravenously into a 23-year-old female volunteer (wt. 47.2 kg, height 5'3") who had been fasted overnight. IOO ml of blood was drawn at I , 2 and 3 h after the injection. The blood was introduced into 700 ml of water and precipitated with IOO ml o.15 M Ba(OH)~ and ioo ml 5~o ZnSO4 (ref. 16). The supernatants from the samples obtained at I and 3 h were passed through a column of Duolite-C-3 in the H + form and Duolite A- 4 in the O H - form (Diamond Alkali Co., Redwood City, Calif., U.S.A.). The effluent and aqueous washings of the column were evaporated to a small volume which was applied to W h a t m a n 3MM paper and chromatographed in the butanol acetic acid-water system. The glucose was isolated, degraded and the degradation products assayed for 14C as described above. The supernatant fluid from the 2-h period was evaporated to about 20 ml, acidified to p H I with o.5 M H2S04, carrier lactate was added and the solution was then continuously extracted with diethyl ether. The diethyl ether extract was neutralized with I M NaOH and evaporated. The lactate in the residue was purified on a celite column ~7 and then degraded and assayed as described above. Biochim. Biophys. Acta, 2Ol (197 o) 425-433
N. FREINKEL e{ al.
430 RESULTS
The distribution of 14C on the glucose formed from rnyo-[12-14C]inositol (Table I) by the slices of kidney showed activity primarily in C-I and C-6. There was 4o-8o% as much activity in C-I as in C-6 in the I-h experiments and 25-35% as much activity in the 4-h experiments. There was more activity in C-2-C- 5 in the experiments of 4-h duration. No differences as a function of dietary state or the concentration of glucose in the medium are apparent. The 14C in the glucose from the blood of the normal subject given m3,o-~2J~C ~inositol (Table II) also was primary in C-I and C-6. There was 70 and 85% as much activity in C-I as in C-6 in the glucose from the blood drawn I and 3 11, respectively, after the myo-ii2-14C]inositol injection and greater randomization of nC into C-2 C-5 at 3 h than I h. Lactate isolated from the medium after the incubations of Expts. I and 5 (Table I I I ) contained 14C primarily in C-3 while the lactate from the blood of the normal subject (drawn 2 h after the mvo-!2-14Clinositol administration) has 70% as much activity in C-I as C-3. Glycerol isolated from the phospholipid fraction contained activity primarily in C-3 (Table IV). T A B L E 1I DISTRIBUTION
OF
14C I N
BLOOD
GLUCOSE
FROM
NORMAL
SUBJECT
ADMINISTERED
tllyo-I2-14C I-
INOSITOL
Time after injection (h)
14C in glucose carbon C-Jr C-2 C-3
C-4
C-5
C-6
Recovery (%)
I
35,o 35 .8
2.6 4 .8
5.0 7 .8
51.9 41'8
94.8 l°°'3
3
4.0 5 .2
1. 5 4 .6
TABLE III DISTRIBUTION
O F 14C I N L A C T A T E F O R M E D F R O M m y 0 - [ 2 - 1 4 C ] I N O S I T O L
BY KIDNEY
SLICES AND THE
NORMAL SUBJECT
Source
1~C in lactate carbon
Expt. I Expt. 5 Normal subject
Recovery
c-f
c-2
c-3
(%)
6.0 5.4 36. i
6.6 6.1 12. i
87. 4 88.5 51.8
87.6 98.o i oo.2
T A B L E IV DISTRIBUTION
O F 14C I N G L Y C E R O L F O R M E D F R O M
myo-E2-14CilNOSITOL
Expt. No.
14C in glycerol carbon C-r C-2 C-3
Recovery (°/o)
2 5 6 7*
34.4 5.6 15.3 31-7
lO6.9 112.o** 92.6 96"5
6.3 9.9 9.3 7 .0
59.3 84.5 75.4 61"3
BY RABBIT KIDNEY
SLICES
* I n E x p t . 7 only glycerol w a s d e g r a d e d . Slices were from fed r a b b i t s , i n c u b a t i o n w a s for 3 h a n d was done in a m e d i u m n o t p r e p a r e d w i t h glucose. ** Glycerol was i s o l a t e d from g l y c e r o p h o s p h a t e b y t r e a t m e n t w i t h a l k a l i n e p h 0 s p h a t a s e .
Biochim. Biophys. Acta, 2Ol (197 o) 425-433
431
I N O S I T O L METABOLISM TABLE
V
DISTRIBUTION IN GLUCOSE AND LACTATE FORMED FROM D-[6-14CJFRUCTOSE BY RABBIT KIDNEY SLICES
Dietary state
Period of incubation (o//)
14C in glucose carbon C-t C-2 C-3 C-4 C-5 C-6
Recovery (%)
1~C in lactate carbon C-± C-2 C-3
Recovery (%)
Fed Fasted
I 4
19.3 17.2
66.2 ( l O l . 4 ) * 16.7 (8o-4)*
o.o lO.4
lOl.9 98. 5
4.6 6.6
o.7 3.0
2.1 2.9
i.t 3.3
72.2 67.0
4.8 9-4
95.2 80.2
* R e c o v e r e d i n o s a z o n e in p a r e n t h e s e s .
In the incubations of [6-14C]fructose with rabbit slices (Table V) there was onefourth as much 14C in C-I as C-6 and again 14C was primarily in these carbons. Incorporation of 14C in lactate was primarily in C-3. DISCUSSION
In no instance have we observed a greater activity in C-I than C-6 of glucose as was reported by ANDERSON AND COOTS7. The distributions in glucose we observe are in accord with the metabolism via the glucuronic acid pathway and pentose cycle. These results document in vitro what has been presumed to occur in the kidney from studies in vivo. In the slice experiment (Table I) the incorporation into C-I can be attributed to both the equilibration of the E3-14Clglyceraldehyde 3-phosphate formed via these pathways (see INTRODUCTION) with dihydroxyacetone 3-phosphate and then formation of glucose, and the cleavage of fructose 6-phosphate formed from the myo-E2-14C~inositol into the triose phosphates, their equilibration and then formation of glucose, i.e. : [6- 14C]fructose-6-P ----~[3- 14C]glyceraldehyde-3-P dihydroxyacetone-3-P
[1,6- 14(2 ]fructose-6-P [1,6- 14C ]glucose
The significance of the lesser incorporation into C-I relative to C-6 after 4 h of incubation than after the earlier time periods is uncertain because of the limited number of experiments performed. The extent of incorporation into C-I compared to C-6 would be dependent upon the rate of isomerization of glyceraldehyde 3-phosphate to dihydroxyacetone 3-phosphate relative to the rate of formation of the glucose and these rates could have changed with time. The greater activity in C-2-C-5 at 4 h than at the earlier hours is in accord with increasing randomization with time of the 14C into oxaloacetate and pyruvate via the reactions of the Krebs cycle and the synthesis of glucose from these compounds. The distribution of 1~C in lactate (Table II) is also in accord with such metabolism with the ~4C in C-3 reflecting formation from E3-14C]glyceraldehyde 3-phosphate and the 14C in C-I and C-2 reflecting randomization via the reactions of the Krebs cycle. Since myo-inositol can be synthesized from glucose by kidney slices ~8, the 14C distributions could conceivably be consequent in some degree to the metabolism of 14C-labeled myo-inositol formed from the 14C-labeled glucose in turn formed from the myo-~2-1~CJinositol added as labeled substrate. This contribution was almost certainly negligible in consideration of the dilution of the ~4C of the myo-[2-1*C~inositol during this recycling and the limited amount of conversion of glucose to mD,o-inositoltS. The distributions of I4C in the glucose from the blood of the subject given n(voBiochim. Biophys. Acta, 2Ol (197 o) 4 2 5 - 4 3 3
432
N. FREINKEL et al.
E2-i4C]inositol are similar to those obtained using the kidney slices, though there perhaps was a tendency to greater incorporation into C-I relative to C-6 by the subject. Most probably the glucose in the blood was formed primarily from myo-i2-I4C] inositol without lactate as an intermediate. If the glucose has been formed from [3-14Cllactate one would expect glucose with 14C extensively in C-2-C-5 in accord with the metabolism of the carbons of lactate in the Krebs cycle prior to their conversion to glucose I9. C-I to C-6 ratios when [6-I4C]Iructose was substrate were in general lower than when myo-E2-i4Clinositol was substrate. If in kidney the [6-i4C]fructose was phosphorylated to [6-14C]fructose 6-phosphate rather than [6-14Clfructose 1-phosphate this would be expected since incorporation into C-I from the [6-i4Clfructose could then only occur through cleavage to the triose phosphates and then resynthesis. The data would indicate that much of [6-14Clfructose was phosphorylated to [6-I4Cifructose 6-phosphate since each molecule of [6-14C]fructose 1-phosphate would yield in its further metabolism a molecule of I3-1~C~glyceraldehyde 3-phosphate and this would then favor increased incorporation into C-I. In accord with this the activity ill the rat of the enzyme fructokinase, catalyzing the formation of fructose 1-phosphate, is several-fold lower in kidney than in liver ~°. In liver where the fructose I-phosphate pathway is so active, C-I of glucose formed from E6-i~Clfructose does have 7o-8o% of the activity of C-6 (ref. ii). This is also the case for blood glucose when i6-1~C!fructose is administered to man (J. MARSHALL, J. CRAIG, K. HOSTETLERAND B. R. LANDAU, unpublished observations). The considerable activity in C-I in two degradations of glycerol formed by kidney slices and in the blood lactate from the subject given myo-~2-I4Clinositol remain unexplained. The glycerols would be expected to reflect the distribution in glyceraldehyde 3-phosphate and hence have activity primarily in C-3 and the lactate should be expected to have a distribution similar to that seen in the experiments in vitro. Note added in proof (Received D e c e m b e r i8th, 1969) Since submission of this p a p e r H a n k e s et al. ~1 have reported a s t u d y of the m e t a b o l i s m of myoinositol in h u m a n s . I n three n o r m a l subjects three nonpentosuric kindred of subjects w i t h pentosuria, two h o u r s after a d m i n i s t r a t i o n of myo-F2-a4C] inositol 3I :~ 2 % and 48 Jz 3% of the 14C in blood glucose was recovered in C-I and C-6 respectively, a distribution similar to t h a t in o u r one subject. ACKNOWLEDGMENTS
This study was supported by Research Grants AM-Io627 and AM-Io699 and by Training Grant AM-5o7I from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service, Bethesda, Md., U.S.A. REFERENCES I E. C. CHARALAMPOUS, S, 13UMILLER AND S. GRAHAM, J. Am. Chem. Soc., 80 (1958) 2022. 2 C. F. HOWARD, Jr. AND L. ANDERSON, Arch. Biochem. Biophys. 118 (1967) 332. 3 J. J- BURNS, in D. M. GREENBERG, Metabolic Pathways, Academic Press, New York, 3rd Ed., 1967, p. 405. 4 E. CHAROLLAIS AND T. POSTERNAK, Helv. Chim. Acta, 48 (1965) 280. 5 T. POSTERNAK, The Cyclitols, Holden-Day, San Francisco, 1965, p. 329 . 6 ]3. R. LANDAU, C*. E. BARTSCH AND H. R. WILLIAMS, J. Biol. Chem., 241 (1966) 75 o. 7 L. ANDERSON AND R. H. COOTS, Biochim. Biophys. Acta, 28 (1958) 666.
Biochim. Biophys. Acta, 2Ol (i97o) 425-433
INOSITOL METABOLISM
433
8 R. STJERNHOLM AND E. P. NOBLE, J. Biol. Chem., 236 (1966) 6I 4, 9 N. FREINKEL AND R. M. C. DAWSON, Biochem. J., 81 (1961) 250. IO N. FREINKEL, A. I~. COHEN, R. A. ARKY AND A. E. FOSTER, J. Clin. Endocrinol. Metab., 25 (1965) 76 . i i J. A. MUNTZ, J. Biol. Chem., 243 (1968) 2788. 12 L. W. WHITE AND t3. R. LANDAU, J. Clin. Invest., 44 (1965) 12oo. 13 J. FOLCH, M. LEES AND G. H. SLOANE-STANLEY, J. Biol. Chem., 226 (1957) 497. 14 N. FREINKEL, A. D. LITONJUA AND E. C. SAEF, Nature, 191 (1961) 804. 15 R. YI. C. DAWSON, N. HEMINGTON AND J. B. DAVENPORT, Biochem. J., 84 (1962) 49716 M. SOMOGYI, J. Biol. Chem., 195 (1952) 19. 17 E. H. SWIM AI','p L. O. KRAMPITZ, J. Bacteriol., 67 (1954) 419 • 18 G. ]:-IAUSER AND V. N. FINELLI, J. Biol. Chem., 238 (1963) 3224 . 19 K. Y. HOSTETLER, H. R. WILLIAMS, W. W. SHREEVE AND B. R. LANDAU, J. Biol. Chem., 244 (1969) 2075. 20 R. C. ADELMAN, F. J. BALLARD AND S. V~rEINHOUSE, J. Biol. Chem., 242 (1967) 3360. 21 L. V. HANKES, W. M. POLIBZER, O. TOUSTER, AND L. ANDERSON, Ann. N . Y , Acad. Sci., 165 (I969) 564 .
Biochim. Biophys. Acta, 2Ol (197 o) 425-433