Contribution of the pentose cycle to glucose metabolism by insects

Comp. Biochem. Physiol., 1970, Vol. 37, pp. 143 to 165. PergamonPress. Printed in Great Britain

C O N T R I B U T I O N OF T H E P E N T O S E CYCLE TO G L U C O S E M E T A B O L I S M BY INSECTS* W. CHEFURKA, Y. HORIE t and J. R. ROBINSON Research Institute, Canada Department of Agriculture, London, Ontario, Canada (Received 21 April 1970) Abstract--1. The rates of in vivo conversion of variously labelled glucose, acetate and gluconate to 14CO, by the cockroach (Periplaneta americana) milkweed bug (Oncopeltus fasciatus) and grasshopper (Melanoplus bivittatus) suggest the occurrence of an active pentose cycle and glycolytic-citric acid cycle in these insects. 2. The activity of the pentose cycle in the male and female cockroach was quantitated by two independent methods; one based on 14CO~ yields from the variously labelled glucose and the other based on the degree of randomization of 14C between C-1 and C-3 of glucose residues of glycogen when the pentose cycle was challenged with glucose-2-1~C. The results were consistent in that in the male cockroach about 21 per cent and in the female about 3 per cent of the glucose was metabolized by the pentose cycle. 3. About 50 per cent of the glucose in the male cockroach was metabolized by nontriose pathways. 4. The contribution of the pentose cycle was about 18 per cent in the milkweed bug and 38 per cent in the grasshopper. 5. The labelling pattern of the glucose carbon atoms suggests (a) complete equilibration between glucose-6-P and fructose-6-P in the male but only partial equilibration in the female and (b) that attempts to quantitate the pentose cycle activity through application of equations derived for mammalian tissue suffer interference from the transketolase reaction and from reversal of non-oxidative portions of the pentose cycle. INTRODUCTION EXTENSIVE enzymic studies have implicated glycolysis and the pentose cycle as the major catabolic routes for the metabolism of carbohydrates in insects. In addition, tracer studies utilizing labelled acetate, pyruvate and intermediates of the citric acid cycle have provided additional evidence for the occurrence of the citric acid cycle in insects (Chefurka, 1965). However, attempts to evaluate the relative importance of these pathways in glucose catabolism in vivo are scanty and limited in scope. W a n g and his associates, utilizing the technique of radiorespirometry, have suggested that only 4 - 9 per cent glucose is metabolized by the pentose cycle in the cockroach Periplaneta americana (Silva et al., 1958). Subsequent applications of this technique suggest that the pentose cycle * Contribution No. 372 of the Research Institute, Canada Department of Agriculture. Holder of N.R.C. Postdoctorate Fellowship. Present address : Sericultural Experiment Station, Suginami-Ku Tokyo, Japan. 143

W. CHEFURKA,Y. HORIEand J. R. ROBINSON

144

contributes at most 45 per cent to glucose catabolism in the boll weevil ( L a m b r e m o n t & Bennett, 1966) and 35 per cent in the larvae of the silkworm (Horie et al., 1968). Agosin and his associates found 4-17 per cent pentose cycle activity in various strains of house flies (1966) and 22 per cent in Triatorna (1963) by using the specific activities of fatty acids derived from glucose-6J4C and glucose-U-t4C, a m e t h o d not without drawbacks (Cheldelin et al., 1962). More recently Chefurka (1966) has applied the method of Katz & W o o d (1960) which is based on the fact that the pentose cycle when challenged with glucose-2-t4C randomizes the 14C between C-1 and C-3 of glucose-6-P or its derivative, glycogen. T h e present paper is an extension of our previous report. It compares the radiorespirometric m e t h o d with the C-2 randomization method in an attempt to quantitate the activity of the pentose cycle and to evaluate some of the limitations of each method. MATERIALS AND METHODS

Insects Cockroaches, Periplaneta americana L, were reared on a mixture of Dog Checkers, yeast and sugar at 27°C and 60% r.h. A constant supply of water was provided. Cockroaches of 2-3 months adulthood were used. Two-striped grasshoppers, Melanoplus bivattatus (Say), were reared from eggs collected in the field and supplied by the Research Station, Canada Department of Agriculture, Saskatoon. The grasshoppers were reared to adulthood in an insectary on a diet of lettuce and bran. Milkweed bugs, Oncopeltus faseiatus (Dallas), were reared in the laboratory under the same conditions as the cockroaches, but on a diet of milkweed seeds.

Chemicals D-Glucose-l-l*C (2"7-10"0 mCi/mM*), D-glucose-2-14C (2"37 mCi/mM), D-glucose6-14C (2"0-5'0 mCi/mM), D-glucose-U-a4C (2"14--3"76mCi/mM), acetate-l-l*C (20'0 mCi/ mM) and acetate-2-~4C (15"4mCi/mM) were purchased from the Atomic Energy of Canada Ltd. Glucose-3,4-14C (9"0 mCi/mM) was prepared from 14C-labelled glycogen kindly supplied by Dr. J. Hogg, Queens College, N.Y. D-Fructose-l-l~C (1-16 mCi/mM) and D-fructose-6-14C (0.36 mCi/mM) were purchased from Nuclear Research Chemicals Inc. D-Gluconate-l-14C (3.42mCi/mM) and D-gluconate-6-14C (4"1 mCi/mM) were purchased from Nuclear-Chicago Corp. All other chemicals were of the highest purity available commercially. The labelled sugars were dissolved in distilled water and diluted to give an activity of approximately 2/zCi/ml for injection into the cockroaches and grasshoppers and 10/~Ci/ml for injection into the milkweed bugs. The precise activity was determined by spotting 10/~1 of the labelled sugar on to discs of filter paper of half-inch diameter. These were oxidized with a chromic acid mixture of Thorn & Shu (1951), trapped in appropriate ionization chambers and the amount of 14CO2 was assayed using a 'Model 6000, Dynacon' vibrating-reed electrometer (Nuclear-Chicago) as already described (Robinson & Chefurka, 1964).

Injection procedure All injections were performed with an 'Agla' micrometer syringe fitted with a 30-gauge needle. The volume of solution injected into the insects was determined by their size; 20/zl of labelled sugar/cockroach or grasshopper; 2/A/milkweed bug. Because the site of * Specific activity.

PENTOSE CYCLE I N INSECTS

145

injection influenced the rate and total output of 14CO2, (Ela et al., 1970) the injection procedure was standardized so that all injections were made into the abdomen between the third and fourth sternites on the right side of the midventral line of an unanesthesized insect. Control injections consisted of only distilled water. For each run 5 cockroaches, 5 grasshoppers or 10 milkweed bugs were used.

Measurement of respired 14CO2 Immediately after injection, the insects were placed in a 250 ml Erlynmeyer flask which was connected to a circulating system containing an ionization chamber and the respired 14CO 2 was recorded continuously as already described (Robinson & Chefurka, 1964) for 6 to 12 hours. T h e data were expressed as (a) cumulative yield of 14CO~ from each labelled sugar, calculated as per cent of the total radioactivity injected, (b) as percentage interval of 14C recovery (Tolbert et al., 1958).

Isolation of intermediates Into each of a batch of male cockroaches 0"96/zCi of glucose-UJ4C was injected. At 10 min, 30 min, 60 rain, 2 hr, 4 hr and 6 hr, after injection, five roaches were decapitated and homogenized in a Waring blender in 35 ml of cold 1 N HC104 solution. T h e homogenate was centrifuged for 10 rain at 3000 rev/min. T h e residue was re-extracted with the same volume of cold 1 N HC104. A weighed amount of the residue was assayed for 14C activity by combustion as described above. T h e two supernatants were combined, neutralized with 2 N K O H and left overnight at I °C to precipitate the KC104. After eliminating the KC 1 0 , by centrifugation an aliquot of the acid-soluble fraction was assayed for a4C activity. Glycogen was precipitated from the acid-soluble fraction by addition of 1"5 vol of 95% ethyl alcohol. After isolation of the glycogen, the supernatant was used for analysis of trehalose. T h e crude glycogen was dissolved in water and re-precipitated several times with 1-5 vol of 95% ethyl alcohol. After the final precipitation the glycogen was dissolved in distilled water and aliquots spotted on paper discs were oxidized and 1*C-level was determined as described above. T h e post-glycogen supernatant was concentrated under reduced pressure in a rotary evaporator and passed through Dowex-1 (formate) and Dowex-50 (H +) exchange resins. T h e aqueous effluent was concentrated and aliquots were applied on No. 1 Whatman filter paper. T h e chromatograms were developed three times in butanol-acetic acid-water (4 : 1 : 5) by the ascending method. T h e trehalose and glucose were localized by the silver nitrate method (Trevelyan et al., 1950) or by scanning the paper with a Geiger-Miiller tube. T h e radioactive spots were cut out, oxidized and assayed for x4C.

Degradation of 14C-glycogen Twenty/~1 of glucose-2-x4c (25/~Ci/ml) were injected abdominally into four lots of 10 male and four lots of 10 female cockroaches. After 1 and 6 hr, the cockroaches in the appropriate lot were decapitated, minced into a 0"IN K O H solution and thoroughly homogenized in a glass-Teflon homogenizer (2-3 m m clearance). T h e homogenate was filtered through cheesecloth. After several washings of the residue, the combined filtrates were digested in 30% K O H for 30 rain in a boiling water bath. T h e solid matter was removed by centrifugation and the glycogen in the supernatant was isolated and purified by repeated precipitation with 95% ethanol in the presence of sodium sulfate (Hassid and Abraham, 1957). It was then dialyzed at 2°C for 60 hr against several batches of distilled water, T h e glycogen was hydrolyzed in 0-167 N H2SO4 for 48 hr. T h e resulting solution was made slightly alkaline, decolorized with charcoal and deionized by passing through a cation (Rexyn 102 H +) and anion (Rexyn RG3, O H - ) exchange columns. T h e final aqueous effluent was reduced, under vacuum, to an amorphous residue consisting mainly

W. CHEFURKA,Y. HORIE and J. R. ROBINSON

146

of 14C-glucose. This was then degraded with Leuconostoc mesenteroides (Berstein and Wood, 1957) and the activity of each of the six carbon atoms was determined by Mr. Williams, Department of Biochemistry, Western Reserve University, Cleveland, Ohio.

Calculations (a) Radiorespirometric method. The percentage of glucose metabolized by the pentose cycle was estimated from the yields of 14COa derived from variously labelled sugars by using the relationship Gp =

G1 - G6 x lOO, ( G 1 - G6) + G3,4

(1)

where G1 = cumulative yield of 14CO~ from glucose-l-14C, G6 = cumulative yield of 14COa from glucose-6-14C G3,4 = cumulative yield of 14CO~ from glucose-3,4-a4C. The assumptions underlying the derivation of this equation have been discussed by Silva et al. (1958). All calculations were based on a R T U value (Cheldelin et al., 1962) of 3 hr. The justification for using this value lies in the fact that no evidence for free radioactive glucose could be found in the cockroach after this time suggesting complete catabolism of glucose. (b) C6/C1 14CO2 ratio. This method of qualitatively evaluating the pentose cycle activity was first introduced by Bloom et al. (1953a,b). Although this approach is complicated by many variables (Wood et al., 1963), it will be shown that, at least for the cockroach, the ratio provides a reliable indication of pentose cycle activity. (c) 14C randomization method. This method is based on the fact that the pentose cycle randomizes the 14C between C-1 and C-3 of glucose-6-P or glucose residues of glycogen when challenged by glucose-2-14C (Katz & Wood, 1960). The percentage of glucose metabolized via the pentose cycle (PC) was estimated by the equations (2), (3) and (4) listed in Table 4. The derivation of equations (2) and (3) is given by Wood et al. (1963), who assumed a model of metabolism in which recycling of fructose-6-P to glucose-6-P was complete. Since the extent of recycling in many tissues is unknown or at best incomplete, Landau et al. (1964) derived equation (4) (Table 4) for the estimation of the pentose cycle. It is based on a model in which no assumptions were made as to the extent of equilibration between glucose-6-P and fructose-6-P. By knowing the a4C distribution in the glucose units of glycogen of a tissue preincubated with glucose-2-14C an estimate can also be made of the rate of isomerization of fructose-6-P to glucose-6-P [E-h, see equations (5) and (6), Table 4]. Equations (2), (3) and (4) were also derived on the assumption that the nontriose pathways i.e. pathways that utilize glucose-6-P without yielding triose phosphates, e.g. trehalose and glycogen synthesis, were negligible. Landau et al. (1964) have shown that the randomization of the label in position 2 of glucose was relatively insensitive to the activity of the non-triose pathway especially when the equilibration factor (E-h) between glucose-6-P and fructose-6-P is greater than unity. Hence these equations may be used as if the non-triose pathway activity were zero. RESULTS

Comparative radiorespirometry (a) Conversion o f glucose to C O 2. F i g u r e 1 shows t h e c u m u l a t i v e recovery of x4CO~ a n d Fig. 2 shows the i n t e r v a l rates of recovery of I~CO~ f r o m specifically

PENTOSE

CYCLE

IN

147

INSECTS

labelled glucose substrates that were injected into male and female cockroaches (a), milkweed bugs (b) and grasshoppers (c). All three species showed similar trends in the conversion rates of variously labelled glucose to CO 2 in that the most rapid recovery of 14CO~ was obtained from glucose-3,4-14C followed by glucose2-14C and then from glucose-6-14C. This conversion pattern is consistent with the glycolytic pathway of metabolism according to which the C-3 and C-4 of MALE

FEMALE

404

4 I ~111st"

&

Io-

"6

~o.

z

.~J~Is~

......

~,s ~

1

:>,,

B

r~ 20 ~

40

c

A

J~

If'If

~'~

"

"I G

o

2

4

6

II

Time (hours) FIG. 1. Patterns of cumulative recovery of 14CO~ from male and female (A) cockroaches, (B) milkweed bugs, (C) grasshoppers injected with glucose-3,4-z4C (v ~ , ~ n ) , glucose-l-14C ( ), glucose-2-14C (vvmvvvvvvvvwvvvvvv,), and glucose-6-14C (- i ~ n ~ m).

glucose are converted to the carboxyl carbon of pyruvate, the C-2 and C-5 become the carbonyl carbon and C-1 and C-6 appear as the methyl carbon. Operation of the Krebs cycle would then demand a preferential combustion of the carboxyl carbon of pyruvate to carbon dioxide. Hence the carbon dioxide recovery rates from glucose-3,4:4C peaked within 15 minutes (Fig. 2) while those from glucose-6a4C peaked 30-60 min after injection. The metabolism of these specifically labelled glucose substrates did not, however, go to completion. About 30-40 per cent of glucose-3,4-14C and 10-15 per cent of glucose-6-14C was metabolized to 14CO 2 after 8 hr by these three species (Fig. 1). It is thus evident that less CO~ was derived from those glucose carbons which required more recycling through the Krebs cycle before becoming the carboxyl

148

W. CHEFURKA, Y. HORIE and J. R. ROBINSON

A

A

\ \

Cf ~

'~j

\

f

_B

_B

c~

vo

c

c co

co i

i

:

Time (hours)

Fro. 2. Interval 1~CO2 recovery from male and female (A) cockroaches, (B) milkweed bugs, (C) grasshoppers. Labelled substrates as in Fig. 1.

PENTOSE CYCLE I N INSECTS

149

carbon of pyruvate. Presumably the acetyl carbon was syphoned off into other cellular constituents such as fatty acids and amino acids. If the glycolytic route were the sole pathway for metabolism of glucose the C-1 and C-6 carbons should be equivalent with respect to the amount of l'CO2 recovered, but they are not (Fig. 2). The additional and rapidly respired 14CO~ from glucose-l-14C by the two sexes of the three species suggests a preferential decarboxylation of glucose-lJ4C presumably by the glucose-6-phosphate and 6-phosphogluconate dehydrogenases of the pentose cycle. The rate of metabolism of C-1 of glucose depended on the species. It was highest for the grasshopper, medium in the cockroach and lowest in the milkweed bug (Fig. 2). In the case of the grasshopper, it exceeded the rate of conversion of glucose-3,4-1~C to CO s suggesting that the pentose cycle may be of major significance in this species. One striking metabolic difference between the sexes was the greater metabolism of glucose-2J4C by the female grasshopper and male cockroach. This could arise from a more active randomization of the C-2 of glucose by the transketolase and transaldolase reactions so that it eventually became the C-1 of glucose. The data in Table 3 show that such randomization was active in the male cockroach. (b) Conversion of acetate to COo. The metabolism of acetate-l-14C and acetate-2-14C by male and female cockroaches, (a), milkweed bugs (b) and grasshoppers (c) is seen in Figs. 3-4. The metabolism of both specifically labelled substrates did not go to completion; about 30-50 per cent of the carbons were recovered as 14CO2 (Fig. 3). Hence a sizeable quantity of acetate was diverted into non-oxidizable cellular components. The interval recovery rates of 14CO 2 from all species (Fig. 4) were considerably less for acetate-2-14C than from acetate- 1-14C as substrate. This metabolic sequence is consistent with the operation of the Krebs cycle. The initial recovery from acetate-2-14C showed a broad peak between 15 to 60 minutes followed by a secondary peak or a shoulder. By contrast, the initial recovery rates of 14CO2 from acetate-lJ4C showed a maximum at 15 minutes after administration of the substrate followed by a smaller maximum or a shoulder. The onset time of this secondary burst of metabolism of acetate-lJ4C depended on the species and sex and showed good coincidence with a similar peak of activity of acetate-2J4C as substrate. Presumably this secondary peak of activity reflected the oxidation of secondary substrates initially formed from acetate. The data in Table 1 show a comparison of the ratios of the yields of 1~CO2 for glucose-2-1~C and glucose-6-14C with those of acetate-l-14C and acetate-2J4C. The agreement between the ratios clearly suggests that the C-2 and C-6 of glucose are equivalent to the C-1 and C-2 of acetate respectively, which is understandable if one supposes a metabolism of glucose by the glycolytic pathway. Furthermore, the agreement between the ratios for the female cockroach suggests that randomization and recycling of the glucose carbons was not very extensive. On the other hand, the discrepancy between the ratios for the male cockroach is in keeping with the extensive randomization of C-2 of glucose to C-1 (Table 3) which would

250

CHEFURKA,

W.

Y.

J.

HORIE a n d

MALE

R.

ROBINSON

FEMALE

5o

30 2C

,0i C ~d'°

B

~c ~' w

20

2

4

6

e0

2

4

6

a

Time (hours)

FIG. 3. Patterns of cumulative recovery of I4CO~ from male and female (A) cockroaches, (B) milkweed bugs and (C) grasshoppers injected with acetate-l-a4C ( ) and acetate-2-14C ( - - - - - - ) .

TABLE Z--COMPARISON OF THE RATIOS OF THE YIELDS OF 14CO2 FROM GLUCOSE-2-14C AND GLUCOSE-6-14C WITH YIELDS OF 14COz FROM ACETATE-I-IaC AND ACETATE-2-14C.

Ratio of 14CO2 from

Species

Periplaneta americana Melanoplus bivittatus Oncopeltusfasciatus

Sex

Glucose-2-14C "Glucose-6-14C

Acetate-1-14C Acetate-2-14C

Male Female Male Female Male Female

1"54 1-26 1 "98 1.69 1.29 1"52

1-54 1.34 1 "79 1 '63 1.28 1"54

T h e ratios were calculated from the cumulative I4CO~ recovery curves of Fig. 1 and Fig. 3 after 8 hr.

PENTOSE CYCLE IN INSECTS

i,

151

A

12

i 4

t~"

2

I

0

I

2

3

I

2

3

Time (hours} FIG. 4. I n t e r v a l 14CO2 r e c o v e r y f r o m male a n d female (A) cockroaches, (B) m i l k w e e d b u g s , (C) grasshoppers. L a b e l l e d s u b s t r a t e s as in Fig. 3.

152

W . CHEFURKA,

Y. HOltIE and J. R. ROBINSON

yield additional quantities of a4CO2. This could also explain the discrepancy in these ratios for the male grasshopper. (c) Conversion of gluconate to CO S. The data in Fig. 5 show that both the male and female cockroach actively decarboxylate gluconate-l-a4C. This suggests the presence of an active gluconokinase and 6-phosphogluconate dehydrogenase. 100 90

Gluconole - I -

C 14~'" . . . . . . . .

Gluconole-6-

CI4s

a

o~

~8o - . o - ,,, 4 .al~,,a,~,.r,,.,e t . . . .dl~

/

o6o

/

~5o "6 -5 40 E

/

"E

/

~2o

l

/

/

/

/

i /

I

!

MALE

FEMALE

/

i

/

10

...,,..........~.---~ I

2

3

4

I

I

I

I

5

6

7

8

I

2

3

4

5

6

7

8

Time (hours)

FIG. 5. Patterns of cumulative recovery of z4CO, from male and female cockroaches injected with gluconate-l-z4C (. . . . . . ) and gluconate-6-14C (). At least 80 per cent of the administered gluconate-l-14C was recovered as 14CO~ suggesting very little diversion of gluconate into non-oxidizable cellular components. Similar active conversion of gluconate 14C has been reported by Silva et al. (1959). In view of the active metabolism of gluconate, it is unlikely that the low metabolism of glucose-l-14C by both sexes could be ascribed to a deficiency of 6-phosphogluconate dehydrogenase. It may however be due to a lower glucokinase activity, a rate-limiting glucose-6-phosphate dehydrogenase or a greater affinity for glucose by phosphohexoseisomerase. By contrast, gluconate-6-14C was metabolized very slowly to 14CO2 by both sexes suggesting that the activity of the pentose cycle did not appreciably relocate the radiocarbon into an oxidizable position of glucose as would be expected if for example triose recombination were active. The interval rates of recovery of 14CO 2from gluconate- 1-14C and gluconate-6-14C are presented in Fig. 6. It is clear that although the rates for both sexes peaked at similar times after injection of gluconate-1J4C the size of the peak for the female was almost twice that for the male. By contrast, the interval recovery rates from gluconate-6-14C were low with no evidence of a peak.

PENTOSECYCLEIN INSECTS

153

Incorporation of labelled glucose into other cellular components To assess the fate of the 60-80 per cent of glucose carbons that could not be accounted for as COz, an analysis was made of the distribution of 14C in various cellular fractions. 501

FEMhLE

MALE

~ 30

•Z ' 11 \ l

0

#

O

I

\ ~

.

2

.

.

.

3

.

~ - - n . ~

4

. . . .

5

* . . . .

6

O

? Time ( hours}

1

,

i

3

I

,

,

5

6

FIc. 6. Interval 14COz recovery from female and male cockroaches. Substrates as in Fig. 5. The data in Fig. 7 present a flow chart of the distribution of radioactivity that was introduced into the male cockroach as radioactive glucose. Glucose was rapidly metabolized as seen by its rapid disappearance. Only 26 per cent of the initial radioactivity was recovered as glucose ten minutes after injection. Two hours after injection no free glucose could be detected. This is consistent with the data in Fig. 2. Most of the radioactivity was found in the acid-soluble fraction ; about half of which was accounted for by trehalose. A substantial fraction of the radioactivity (18-25 per cent) was also incorporated into the acid insoluble components. Significantly, only a small fraction of the glucose carbons (about 3 per cent) were incorporated into glycogen. Thus about 50 per cent of the radioactivity was found in compounds derived from non-triose phosphate pathways which utilize glucose-6-P to yield such products as glycogen, trehalose, and chitin but not triose phosphates.

Estimation of pentose cycle (a) C J C 1 ratios. A time course plot of the C6/C 1 ratios for both sexes of the cockroach, milkweed bug and grasshopper is presented in Fig. 8. The data show that in all cases, the C6/C 1 ratios were less than unity suggesting a preferential decarboxylation of CO2 from C-1 of glucose as compared with that

80-

0

I

2

4

Time (hours)

:5

5

x~

•

6

7

14C02 (0), trehalose (x), glucose (®), acid-soluble fraction (~1,) and acid-insoluble fraction (A).

FIG. 7. Time-course distribution pattern of 14C in glycogen (11),

I0

20

~5~4oll

¥

60-

o 70-

i

-,

90"

I00-

0

t 0

I

2

4

5

Time (hours)

3

6

4,1~i.,e~Js~4wl

americana

Melanoplus bivittatus

~.~4~,.~.~ ~

Periplaneta

C 6 / C 1 ratio as a f u n c t i o n

~B j

J

FIG. 8.

0.2- ~

0.4-

02"

C, 0.4'

C_~s o6

0.8"

0.2"

04"

0.6"

08-

~.0"

of t i m e .

7

pis.mlw4

8

©

.=

O

>

C~

4~

155

PENTOSE CYCLE IN INSECTS

of C-6 presumably by the pentose cycle. In all instances the ratios showed a time-course shift toward unity but only in the case of female cockroaches did it achieve unity. A similar shift in C6/C1 ratios has been recently reported for silkworm larvae (Horie et al., 1968) and for plants (Daly et al., 1961). The reason for this shift is at present not clear. It could result from a gradual utilization of endogenous substrates. Certainly, one would expect that the longer an experiment of this type was allowed to run, the more complete would be the utilization of endogenous substrate and hence the C6/C 1 ratio would approach unity as a function of time. This however could hardly provide the entire explanation because in the cockroach, the glucose reserves were exhausted within 2 hr. Furthermore, trehalose, a potential reserve, accumulated and remained at a steady state for the duration of the experiment. This shift also suggests that the C6/C 1 ratio sampled at a particular time during the experiment was not necessarily a reliable qualitative index of the activity of the pentose cycle. As will be seen later, there was better consistency between the early ratios and the 14C randomization experiments as an index of pentose cycle activity. The slight pentose cycle activity as indicated by the early C6/C1 ratios in the female cockroach would have been overlooked had reliance been placed only on the later ratios. As has been discussed on numerous occasions, the C6/C 1 ratio though useful for the detection of pathways, suffers from serious limitations for pathway evaluation. At best it provides a qualitative indication of pentose cycle activity (Wood, 1960; Cheldelin et al., 1962). Perhaps one of its most serious limitations is the possibility of preferential dilution of the labelled carbon atoms of the substrate by endogenous carbon reserves. (b) Radiorespirometry method. It is possible to estimate the percentage of glucose oxidized to CO2 by the pentose cycle by employing the yield of 14CO2 from glucose-6-x4c and glucose-l-14C at an R T U of 3 hr (Fig. 2). As discussed by Cheldelin et al. (1962), yield data are not affected by possible differential rates of metabolism of substrates nor by dilution by endogenous carbon pools. The data in Table 2 show the extent of participation of the pentose cycle in the catabolism of glucose estimated by equation (1). Negligible activity (2.4 per cent) TABLE2--PERCENTPENTOSECYCLEIN INSECTS Species Periplaneta americana Oncopeltusfasciatus Melanoplus bivittatus

Sex

Gp(%)

Male Female Male Female Male Female

16"7 2'4 13'4 16"4 38'7 39-9

T h e cumulative yields of a4CO2 were obtained from Fig. 1 at an R T U of 3 hr.

156

W . CHEFURKA, Y. HORIE a n d

J.

R. ROBINSON

is indicated by these data for the female cockroach. About 13-17 per cent of the glucose was catabolized via the pentose cycle in the male cockroach and the male and female milkweed bug and about 40 per cent in both sexes of the grasshopper. Silva et al. (1959) have suggested that the glycolytic-citric acid cycle activity (GE) may be calculated by GE = 100--G~. This relation, which is valid only if no additional pathways are available for glucose catabolism, is an invalid assumption in view of the high activity (about 50 per cent) of the non-triose pathways in the male cockroach (Fig. 7). Thus it appears that in v i w only about 50 per cent of the glucose was metabolized by the pentose cycle and the glycolytic-citric acid pathway in the male cockroach. Since the pentose cycle metabolized about 17 per cent of the glucose, then about 33 per cent of the glucose was metabolized by the glycolytic-citric acid cycle. (c) Randomization of glucose-2-11C. Katz & Wood (1960) have presented a method for estimating the activity of the pentose cycle by quantitating the randomization of 1~C of labelled glucose-2-14C in glucose-6-P or in glucose units of glycogen. The flow of label during this randomization process which is catalyzed by the transketolase and transaldolase reaction is shown by reactions (2) and (3). The data in Table 3 show the distribution of the ~C in the glucose residues of glycogen isolated from male and female cockroaches one and six hours after injection with glucose-2-14C. Active randomization of C-2 into C-1 and C-3 is TABLE 3--DISTRIBUTION OF 14C IN GLYCOGEN AFTER INJECTION OF OLUCOSE-2-14C

Periplaneta

WITH

Specific activity in carbon atoms of glucose unit* Time (hr)

Sex

1

2

3

4

5

1 6 1 6

Male Male Female Female

31 "4 81 '1 4.1 5'9

100 100 100 100

16-9 37"7 5.0 7"1

1"4 1-6 0"5 0-28

7"8 7-9 12"1 7-1

6 2'2 3'5 1-1 0.22

Recovery (%) 86 94 61 93

* Relative specific activity in individual c a r b o n atoms of glucose f r o m glycogen with the c a r b o n - 2 activity set to 100. T h e p e r c e n t a g e recovery = v activity of individual carbon atoms " activity of glucose totally oxidized x 100.

evident indicating pentose cycle activity in the male cockroach. The extent of this randomization was time-dependent. As is evident from Table 3, about 31 and 81 per cent of the injected glucose-2-14C activity was detected in C-1 at one and six hours after injection. By contrast to the male, the data for the female show low randomization of a4C, suggesting low pentose cycle activity. Furthermore, the randomization did not increase with time as dramatically as it did in the male. About 5-6 per cent

PENTOSE CYCLE IN INSECTS

157

of the initially injected glucose-2-14C activity was detected in C-1 at one and six hours. Several additional points are worthy of note. Firstly, the specific activity of C-1 was more than double that of C-3 in the male between 1 hour and six hours. This preponderance of activity in C-1 probably reflects incorporation via the transketolase reaction as follows: pentose-5-P,- 1-14C + erythrose-4-P -+ fructose-6-P,-1-14C + glyceraldehyde-3-P.

(1)

This label in fructose-6-P,-1-14C when mixed with the 1,3 label formed via the cycle in the oxidative direction, i.e. 3 glucose-6-P,-2-14C-+ 3 CO2+ 3 pentose-5-P,-1-14C,

(2)

2 pentose-5-P,-1-14C -+ fructose-6-P,-1,3J4C + erythrose-4-P,

(3)

would yield a pool of hexose with the specific activity of C-1 twice that of C-3. A consideration of reactions (1), (2) and (3) suggests that the pentose cycle in the male cockroaches may be regarded as catalyzing 3 glucose-6-P -+ 3 COs + 2 fructose-6-P + 1 glyceraldehyde-3-P. As will be shown later, recycling of fructose-6-P appears to be complete so that 2 fructose-6-P ~- 2 glucose-6-P. Consequently the pentose cycle in the male cockroach could be formulated as follows: glucose-6-P -+ 3 CO 2+ glyceraldehyde-3-P. It is thus evident that erythrose-4-P formed by reaction (3) would not accumulate because it would be used up in the transketolase-catalyzed reaction (1). Hence the major end-product of the pentose cycle in the male roach is glyceraldehyde-3-P. By contrast with the male, the transketolase-catalyzed reaction (1) was low or absent in the female cockroach. Instead, a small but consistently higher specific activity of C-3 over that of C-1 was evident. This suggests incorporation via partial reversal of the non-oxidative portion of the cycle as follows: fructose-6-P,-2-14C + glyceraldehyde-3-P -~ pentose-5-P,-2A4C + erythrose-4-P,

(4)

2 pentose-5-P,-2-14C -+ fructose-6-P,-2,3-14C + erythrose-4-P.

(5)

Fructose-6-P,-2,3J4C could then mix with label 1,3 formed via the cycle in the oxidative direction as formulated by reactions (2) and (3) to yield more label in C-3 than in C-2. A consideration of reactions (2), (3), (4) and (5) clearly suggests the accumulation of erythrose-4-P as the major end product of the pentose cycle in the female cockroach. Since, as will be shown later, there was extensive isotopic equilibration between glucose-6-P and fructose-6-P, the 1 hr C1/C 2 and C3/C 2 ratios when substituted in equations (2) and (3) (Table 4) give calculated pentose cycle of 22-8 and

158

W. CHEFURKA,Y. HORIEand J. R. ROBINSON

20"5 per cent respectively. On the other hand the estimate of pentose cycle based on a model in which no assumptions were made as to recycling[equation (4)] is 21.5 per cent. Clearly, good agreement was obtained regardless of the basic assumptions regarding recycling. Furthermore, these estimates agree with that by the radiorespirometric method. The C3/C 2 ratio taken six hours after administration of glucose-2J4C gives a pentose cycle value of 60 per cent while the six hour C1/C 2 ratio yields an absurd value of 214 per cent. The estimate by equation (4) yields an equally unrealistic value of 377 per cent. This discrepancy between one hour and six hour values may be attributed to reaction (1). This reaction could contribute sizeable quantities of fructose-6-P,-1-14C to the hexose phosphate pool and so distort the relative specific activity of C-1 over that of C-3. The effect of this interference may be reduced by employing the C6/C 5 and C4/C s ratios for the estimation of the pentose cycle. The validity for this stems from the activity of the so-called "pick-up" reaction (Landau & Bartsch, 1966) in which glycerladehyde-3-P generated by the pentose cycle enters the pool of triose phosphate from which a molecule of glyceraldehyde-3-P then re-enters the pentose cycle, via

glyceraldehyde-3-P + sedoheptulose-7-P ~ fructose-6-P + erythrose-4-P,

(6)

to provide carbon atoms 4,5,6 of fructose-6-P which is isomerized to glucose-6-P. Therefore carbon atoms 6,5 and 4 of glucose-6-P should reflect the relative distributions of 14C of carbons 1,2 and 3 of glucose-6-P (Landau & Bartsch, 1966). This is evident from Table 3 which shows that the one hour C-1 : C-2 : C-3 ratio for males is 0-314 : 1 : 0.169 and the C-6 : C-5 : C-4 ratio was 0-286 : 1 : 0.174. It is clear that they are proportional even though much greater activity was present in the upper than lower carbon atoms. Substituting the C6/C 5 and C4/C 5 for C1/C ~ and C3/C 2 in equations (2) and (3) a value for the pentose cycle of 20 and 21 per cent is obtained. For the six-hour experiment the C-1 : C-2 : C-3 ratio is 0.811 : 1 : 0.377 whereas the C-6 : C-5 : C-4 ratio is 0.433 : 1 : 0-203. Clearly these ratios are not proportional. However, the six-hour C-6 : C-5 : C-4 ratio is in reasonable agreement with the one-hour C-1 : C-2 : C-3 ratio. Hence by substituting this Ce/C 5 ratio for C1/C 2 and C4/C 5 for C3/C 2 a pentose cycle activity of 38 and 25 per cent is obtained (Table 4). Likewise when Ce/C 5 was substituted for C1/C~ in equation (4) a value of 41 per cent is obtained. Consistency between the one-hour and six-hour activities is therefore much improved. The CG/C5 value probably represents the maximal while the C4/C 5 ratio, the minimal value for the pentose cycle. The C1/C 2 and C3/C 2 ratios [equations (2) and (3), Table 4] for the female cockroach give a calculated pentose cycle activity of 2.1 and 2.6 per cent at one hour and 3.1 and 7.6 per cent at six hours. The increase in the calculated activity at six hours probably results from a slight distortion of labelling of carbon-3 by reactions (4) and (5). Estimate of the pentose cycle by equation (4) yields a value of 4.4 and 1.6 per cent for 1 hr and at 6 hr. This consistency between equations (2) and (3) and equation (4) also suggests that the pentose cycle activity in both sexes

159

PENTOSE CYCLE IN INSECTS TABLE 4

E S T I M A T I O N OF P C AND

E_n FROM RANDOMIZATION OF Periplaneta

GLUCOSE-2-14C

INTO GLYCOGEN OF

Female

Male 1 hr

6 hr

1 hr

6 hr

0-314 0.169 0-282 14.32

0.811 0.377 0.443 23-15

0.041 0.050 0.091 3.73

0.059 0.071 0.031 26'8

(2)

0.228

0-021

0'031

(3)

0.205

2-14 (0.38) 0.605 (0.25)

0.026

0.076

3.77 (0.411)

0.044

0.016

CI/C~ C3/C2 C6/C5 C1/C6 Calculations: C1/Cz = PC/(1 + 2PC) Cz/C~ = PC/(1 + P C )

PC =

[2 + (C1/C2)] CJC5 [6 - (5C1/C2)] C6/C5 + [ 2 - CffC2] [ 2 - (3Cx/C2)]

{ [ 2 - (C,/C=] + 2Ce/C5} Cx/Cz E_h = [(C6/C5) _ (C1/Ci)] [ 2 - (C,/C,)]

C1 C~

2PCE_h E-h + (1 -- PC - NTP) + 2PCE_h

(4)

0.215

(5)

13.1

(6)

7"3

1-0

* Equations (2) and (3) are from Wood et al. (1963) and equations (4), (5) and (6) are from Landau et al. (1964). Y1/Y~ in the original equation (5) was replaced by CJC5 because Yx/Y2 = C6/C5 when recycling is complete (Landau & Bartsch, 1966). N T P (non-triose pathway) in equation (6) was assessed to be 50 per cent (see text). of the cockroach was not significantly affected by recycling. It was of interest therefore to estimate the extent of recycling in the male and female cockroach.

Degree of isotopic equilibration of hexose phosphates T h e equilibration of fructose-6-P and glucose-6-P can be evaluated by comparing the rates of release of 14CO 2 f r o m C-1 and C-6 of glucose-14C and fructose-14C or by application of equations (5) and (6) (Table 4) derived by L a n d a u et al. (1964). I f fructose-6-P formed by the pentose cycle were completely interconvertible with glucose-6-P t h e n the C , / C 1 ratio measured with fructose-14C would be similar to that measured with glucose-14C. I f on the other hand, isotope equilibration between b o t h hexose phosphates was incomplete then the ratios measured with fructose-14C would be expected to be smaller because a substantial portion of the fructose generated by the pentose cycle would be metabolized glycolytically. T h e recovery of 14CO~ as a function of time is presented in T a b l e 5. Both the male and female cockroach metabolized fructose very slowly. Only 6 - 7 per cent of fructose-laC was converted to 14CO 2 over a f o u r - h o u r period.

W. CHEFURKA,Y. HORIEand J. R. ROBINSON

160

This could be due to a slower penetration of tissues by fructose and/or from low fructokinase activity. T h e C J C 1 ratios derived from glucose as substrate (Fig. 1) are also presented in Table 5. T h e data in Table 5 show that in the case of the male the Ce/C 1 ratios from glucoseJ4C and fructose-14C were in the range 0.60-0.70. This suggests that recycling of fructose-6-P in the pentose cycle was probably unrestricted hence its equilibration with glucose-6-P was nearly TABLE 5 - - 1 4 C O 2

PRODUCTION BY Periplaneta americana INJECTED WITH LABELLED GLUCOSE-14C AND FRUCTOSE-14C

SPECIFICALLY

Radioactivity ('}'0) recovered as I~CO~ from Glucose-14C Duration of exp. (hr) 2 3 4

Fructose-14C

Sex

C-1

C-6

C8/C1

C-1

C-6

C6/C1

Female Male Female Male Female Male

11"62 15.48 13'65 18"45 15.10 20"50

10-05 9'68 12"74 12"07 15'01 13"80

0"87 0"62 0"93 0"65 1"0 0"67

4"24 1"46 5.49 2"81 7'02 4.71

2"89 1"14 4'32 1"83 5"92 2'88

0'84 0"78 0'79 0'65 0.84 0"61

Each cockroach was injected with 20/xl of fructose-l-laC or fructose-6-14C as described in the text. Five cockroaches were used per run. The results given are averages of two to four experiments. complete in the male. This is confirmed by the E h value of 7.3-13.1 [equations (5) and (6), Table 4] which suggests that the rate of isomerization of glucose-6-P to fructose-6-P was seven to thirteen times the rate of glucose utilization. By contrast, the CG/C 1 ratio from fructose was about 0-8 while that from glucose was about 1.0 for female cockroach suggesting incomplete equilibration. This is again confirmed by an E_ h value of about 1.0. DISCUSSION T h e radiorespirometric data provide patterns which reflect the sequential release of the glucose carbons as it undergoes oxidative degradation. If glucose were metabolized only by the glycolytic process, carbons-1 and -6, -2 and -5, -3 and -4 would be equivalent in that the carbons-1 and -6 become the methyl, carbons-2 and-5 the carbonyl and carbons-3 and -4 the carboxyl carbon ofpyruvate. Oxidative decarboxylation of pyruvate would preferentially yield CO 2 from carbons-3 and -4 and an acetate unit. In the latter, the carboxyl and methyl carbons would correspond to the original carbons-2 and -6 of glucose respectively. T h e fact that carbons-3 and -4 of glucose gave the highest yield of ~4CO2 followed by carbon-2 and then carbon-6 is in line with this reasoning and suggests that a substantial portion of glucose was metabolized by the glycolytic pathway in the three species of insects that were studied. Furthermore, the preferential combustion of the

PENTOSE CYCLE IN INSECTS

161

carboxyl carbon of acetate to carbon dioxide by the three species suggests the operation of Krebs cycle. Further confirmation of the participation of glycolysis and the Krebs cycle in the dissimilation of glucose comes from the similarity of the ratios (Table 2) when the yields of 14CO~ from glucose-2-14C and glucose-6J4C are compared with those of acetate-l-14C and acetate-2-14C. This study also shows that carbons-1 and -6 of glucose were not equivalent because carbon-1 was preferentially oxidized to CO s over that of carbon-6 by all the insects studied. This non-equivalence can be taken as indirect evidence for the participation of the oxidative portion of the pentose cycle, and the extent of non-equivalence presumably reflects the activity of this portion of the pentose cycle. The randomization of carbon-2 of glucose provides insights primarily into the nature of the non-oxidative reactions of the pentose cycle. Thus the distribution of the label in the glucose unit of glycogen isolated after a period of randomization suggests the presence of the normal transketolase- and transaldolase-catalyzed reactions: 2 pentoses-5-P -~ sedoheptulose-7-P + glyceraldehyde-3-P, sedoheptulose-7-P + glyceraldehyde-3-P ~ fructose-6-P + erythrose-4-P. These reactions have also been established in other insect species (see review Chefurka, 1965). In addition, two unusual labelling patterns were observed. In the male, the specific activity of carbon-1 exceeded that of carbon-3 by a ratio of 2 : 1. This suggests that erythrose-4-P may serve as an acceptor of the ketol group donated by xylulose-5-P thus yielding another molecule of fructose-6-P and glyceraldehyde3-P as follows: pentose-5-P + erythrose-4-P -~ fructose-6-P + glyceraldehyde-3-P. The net result of these reactions is that the overall pentose cycle in the male may be formulated as follows: 3 glucose-6-P ~ 3 CO2 + 2 fructose-6-P + 1 glyceraldehyde-3-P. Randomization of glucose-2-14C by the female cockroach yielded an excess of label in carbon-3 over carbon-2. This indicates a partially reversed transaldolase reaction in which fructose-6-P serves as a donor of the dihydroxyacetone group to the acceptor glyceraldehyde-3-P and a reversed transketolase reaction in which two molecules of pentose-5-P generate a molecule of fructose-6-P and erythrose-4-P. As a result, erythrose-4-P becomes one of the major end products of the pentose phosphate cycle in the female cockroach. If the pentose cycle were the sole means of randomization of label in carbon-2 of glucose, no label should be detected in carbons-4, -5 and -6. The occurrence of label in carbons-4, -5 and -6 which mirrors the distribution of carbons-l, -2 and -3 suggests the presence of a mechanism that can direct label from carbon-2 7

162

Y. HORIE,W. CHEFURKAand J. R. ROBINSON

into these positions. This can occur by (a)the transaldolase "pick-up" reaction (Landau & Bartsch, 1966), (b) the transaldolase exchange reaction (Landau & Bartsch, 1966), and (c) the condensation of dihydroxyactone-P with glyceraldehyde3-P to form fructose-l,6-diP and then fructose-6-P. The relative importance of these reactions in the relocation of label from carbon-2 to carbons-4, -5 and -6 is not yet known. With the data derived from the radiorespirometric studies and from the glucose-2-14C randomization studies, an estimate of the extent to which the pentose cycle participates in the degradation of glucose has been made. Both methods were remarkably consistent when applied to Periplaneta. Both estimates suggest that approximately 3 per cent of the glucose was metabolized by the pentose cycle in the female and about 16 to 20 per cent in the male cockroach. This is a higher estimate for the male than that provided by Silva et al. (1958, 1959). The discrepancy may be related to the fact that Silva et al. used starved male cockroaches. As will be shown in another communication (Ela et al., 1970) the nutritional state of the insect affects the activity of these metabolic pathways. In the milkweed bug, the pentose cycle degraded 13-16 per cent of the glucose while in the grasshopper its participation was about 38-40 per cent. These quantitative estimates support a pentose cycle activity based on the low C6/C 1 ratios. The surprising consistency of these results may be fortuitous in the face of the potential sources of error which beset the calculations of the pathways of glucose metabolism in vivo. We must therefore emphasize caution in the interpretation of these results and point out that this study is a forerunner to a detailed in vitro investigation of certain metabolically active tissues of these insects. On the other hand, this consistency suggests the possibility of a certain amount of leeway with respect to the rigorousness of the assumptions underlying the model of metabolism assumed in these estimates. One set of equations used in the estimation of pentose cycle activity is based on a model of metabolism that assumes total equilibration and unrestricted recycling of hexose phosphates, no synthesis of fructose-6-P from triose phosphate and irreversible transketolase and transaldolase reactions (Landau et al., 1964; Landau & Bartsch, 1966). Recycling describes a situation in which fructose-6-P, generated by the pentose cycle is converted to glucose-6-P which re-enters the cycle. At present, no information is available to evaluate the extent to which recycling is a general feature of the pentose cycle in insects. Recent studies with mammalian cells indicate that under certain conditions, recycling is complete (Merlevede et aL, 1963; Weaver & Landau, 1963; Lowry & Passoneau, 1964; Rose & O'Connell, 1964) though restricted recycling has also been reported (Landau & Katz, 1954). The equivalence of the Ce/C 1 ratios of the 14CO 2 yields derived from both glucose-14C and fructose-14C suggests complete recycling and equilibration of hexoses in the male cockroach. The non-equivalence of those ratios in the case of the female suggests incomplete equilibration. This conclusion is confirmed by estimates of E_ h. It is worth recalling however that similar estimates of pentose cycle activity in the cockroach were obtained when a model

PENTOSE CYCLE IN INSECTS

163

of metabolism was employed which made no assumption as to the extent of recycling. It is evident therefore that the extent of recycling does not affect seriously the estimates of the pentose cyclein the cockroach. Similar considerations apply to the interference by non-triose pathways. Landau et al. (1964) showed that the activity of the non-tHose pathway did not seriously affect the estimate of the pentose cycle activity. Hence for all practical purposes the glycolyticpathway need not be differentiated from the non-triose pathway. This insensitivity of the pentose cycle to the activity of the non-triose pathway becomes greater as isomerization is more complete. The present study does however, emphasize that reversal of transketolase and transaldolase reactions could introduce serious errors into the estimates of pentose cycle activity. These reactions significantly distort the distribution of a4C in C-I and C-3 of glucose from what is predicted by the model of the pentose cycle. This distortion exaggerates the C,/C 2 and Ca/C2 ratios and hence prevents accurate determination of the contribution of the pentose cycle. It is not surprising, therefore, that this method has enjoyed success primarily with systems where the specific activity of C-l and C-3 were approximately equal and the pentose cycle activity was low. Landau & Katz (1964) have suggested that such reversed transketolase and transaldolase reactions may account for the discrepancy in the estimate of the pentose cycle by the C1/C 2 and C3/C 2 ratios for rat adipose tissue treated with insulin. However, reliable estimates of pentose cycle activity were obtained by using C6/C 4 and C5/C 4 ratios instead of the CI/C 2 and C3/C 2. Presumably by a transaldolase "pick-up" reaction (Landau & Bartsch, 1966) and/or the transaldolase exchange reaction some of the activity of the upper half of the hexose molecule is relocated into the lower half in a proportional manner. The fact that the activity of C-6 and C-4 relative to C-5 was not distorted to the same extent as that of C-1 and C-3 suggests the occurrence of two pools of hexose phosphates (Landau & Sims, 1967) only one of which may provide triose phosphates for pick-up and/or exchange reaction. It would appear then, that the model of the pentose cycle assumed in the derivation of the equations of Katz and Wood as well as those of Wang and his colleagues has general application. Certain theoretical restrictions imposed on the use of either model are either insignificant or can be side-stepped to yield consistent results when applied to insects. This is at least one decisive test of confidence that the model of metabolism probably approximates physiological reality. Acknowledgements--We wish to thank Miss J. Brown, Mr. S. J. Bajura, Mr. H. Rode, Mr. H. J. Murphy and Mr. J. I. Pullen for technical assistance.

REFERENCES AGOSIN M., FINE B. C., SCARAMELLIN., ILLIVICKYJ. & ARAVENDAL. (1966) The effect

of DDT on the incorporation of glucose and g]ycine into various intermediates in DDT-resistant strains of Musca domestica L. Comp. Biochem. Physiol. 19, 339-349.

164

W. CHEFURKA,Y. H o m e and J. R. ROBINSON

AGOSlN M., SCARAMELLI N., DINAMARCA M. L. & ARAVENDA L. (1963) Intermediary carbohydrate metabolism in Triatoma infestans (Insecta; H e m i p t e r a ) - - I I . The metabolism of 14C-glucose in Triatoma infestans nymphs and the effect of D D T . Cutup. Biochem. Physiol. 8, 311-320. BERSTEIN I. A. & WOOD H. G. (1957) Determination of isotopic carbon patterns in carbohydrate by bacterial fermentation. In Methods of Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol 4, pp. 561-584. Academic Press, New York. BLOOM, B. & STETTEN, D. (1953) (a) Pathways of glucose catabolism. 07. Am. Chem. Soc. 75, 5446. BLOOM, B., STETTEN, M. R. & STETTEN, D. (1953) (b) Evaluation of catabolic pathways of glucose in mammalian systems. 07. biol. Chem. 204, 681-694. CHEFURKA, W. (1965) Intermediary metabolism of carbohydrates in insects. In The Physiology of Insects (Edited by ROCKSTEIN, M.), Vol. II, pp. 581-667. Academic Press, New York. CHEFURKA, W. (1966) Estimation of pathways of carbohydrate metabolism in insects. Proc. entomol. Soc. Ont. 96, 17-23. CHELDELIN V. H., WANG C. H. & KING Z. E. (1962) Saccharides. Alternate routes of metabolism. In Comparative Biochemistry (Edited by FLORKIN M. & MASON H. S.), Vol. 3, part A, pp. 427-502. Academic Press, New York. DALY J. M., BELL A. B. & KRUPKA L. R. (1961) Respiratory changes during development of rust diseases. Phytopathology 51,461-471. ELA R., CHEEURKAW. & ROBINSON J. R. (1970) Glucose metabolism in normal and poisoned Periplaneta americana L. in vivo. 07. Insect Physiol. (In press.) HASSlD W. Z. & AmtAHAM S. (1957) Chemical procedures for analysis of polysaccharides. In Methods of Enzymology (Edited by COLOWlCK S. P. and KAPLAN N. O.), Vol. III, pp. 34-50. Academic Press, New York. HOmE Y., NAKASONE S. & ITO T. (1968) T h e conversion of 14C-carbohydrates into CO2 and lipid by the silkworm Bombyx mori. 07. Insect Physiol. 14, 971-981. KATZ J. & WOOD H. G. (1960) T h e use of glucose-C 14 for the evaluation of the pathways of glucose metabolism. ~7. biol. Chem. 235, 2165-2177. LAMBREMONT E. N. & BENNETT A. F. (1966) Lipid biosynthesis in the boll weevil. Formation of acetate precursor for lipid synthesis from glucose and related carbohydrates. Can.07. Biochem. 44, 1597-1606. LANDAU B. R. & BARTSCH G. E. (1966) Estimation of pathway contributions to glucose metabolism and the transaldolase reactions. 07. biol. Chem. 241, 741-749. LANDAU B. R., BARTSCH G. E. KATZ J. & WOOD H. G. (1964) Estimation of pathway contributions to glucose metabolism and the rate of isomerization of hexose-6-phosphate. 07. biol. Chem. 239, 686-696. LANDAU B. R. & KATZ J. (1964) A quantitative estimation of the pathways of glucose metabolism in rat adipose tissue in vitro. 07. biol. Chem. 239, 697-704. LANDAU B. R. and SIMS A. !NT.(1967) On the existence of two separate pools of glucose-6phosphate in rat diaphragm. 07. biol. Chem. 242, 163-172. LOWRY O. H. & PASSONEAUJ. V. (1964) The relationships between substrates and enzymes of glycolysis in brain. 07. biol. Chem. 239, 31-42. MERLEVEDE W., WEAVER G. & LANDAU B. R. (1963) Effect of thyrotropic hormone on carbohydrate metabolism in thyroid slices. J. clin. Invest. 42, 1160-1171. ROBINSON J. R. & CHEFURKA W. (1964) Continuous measurement of C1402 respired by insects. An ionization chamber method. Anal. Biochem. 9, 197-203. ROSE I. A. & O'CONNELL E. L. (1964) T h e role of glucose-6-phosphate in the regulation of glucose metabolism in human erythrocytes. 07. biol. Chem. 239, 12-17. SILVA G. M., DOYLE W. P., & WANG C. H. (1958) Glucose catabolism in the american cockroach. Nature, Lond. 182, 102-104.

PENTOSE CYCLE I N INSECTS

165

SILVA G. M., DOYLE W. P. & WANG C. H. (1959) Glucose catabolism in the D D T treated american cockroach (Periplaneta americana L.). Arq. Portugueses de Bioq. 3, 298-305. THORN J. A. & SHU P. (1951) A new apparatus for rapid carbon determination by wet combustion. Can. J. Chem. 29, 558-562. TOLBERT B. M., LAWRENCE J. H. & CALVIN M. (1955) Respiratory carbon-14 pattern and physiological state. In Prac. Int. Conf. Peaceful Uses of Atomic Energy, Geneva, Vol. 12, pp. 281-285. TREVELYAN W. E., PROCTER D. P. ~; HARRISON J. S. (1950) Detection of sugars on paper chromatograms. Nature, Lond. 166, 444-445. WEAVER G. ~: LANDAUB. R. (1963) Contribution of the pentose cycle to glucose metabolism by adrenal cortex in vitro. Endocrinology 73, 640-646. W o o d H. G. (1960) Significance of alternate pathways in the metabolism of glucose. Physiol. Rev. 35, 841-859. W o o d H. G., LATZ J. & LANDAU B. R. (1963) Estimation of pathways of carbohydrate metabolism. Biochem. Z. 338, 809-847.

Key Word Index--Pentose pathway in insects; Periplaneta americana; Oncopeltus f asciatus ; Melanoplus bivittatus ; glucose metabolism.