Regulation of Hyalophora cecropia fat body hexokinase by hexose phosphates common to the pathways of glycolysis, glycogen and trehalose synthesis

Comp. Biochem. Physiol., 1976, Vol. 5311, pp, 405 to 413. Peroamon Press. Printed in Great Britain

REGULATION OF H YALOPHORA CECROPIA FAT BODY HEXOKINASE BY HEXOSE PHOSPHATES COMMON TO THE PATHWAYS OF GLYCOLYSIS, GLYCOGEN AND TREHALOSE SYNTHESIS ARTHUR M. JUNGREIS

University of Tennessee, Department of Zoology, Knoxville, TN 37916, U.S.A.

(Received 1 November 1974)

Almtraet--1. Hexokinase from Hyalophora cecropia fat body has been partially purified and characterized. 2. Hexokinase has a broad band of activity between pH 7-0-8.8, with an optimum at pH 8-4. 3. Employing sucrose density gradient centrifugation of a partially purified preparation, a preliminary tool. wt of 64,000 was obtained. 4. Larval and pupal fat body hexokinase had--toward glucose--identical Km's (1.3 x 10-3 M), and Ki's (3.5 x 10-3 M) for G1P. 5. The enzyme is non-competitively inhibited by G6P and F6P (Ki's = 3.0 x 10-4M and 4"0 x 10-*M), and un-competitively inhibited by ADP and AMP (K: = 1-5 x 10-3 M and 0"9 x 10-2 M). 6. F1P, F16P, ATP T6P, UDP-Glucose, Trehalose and 3',5'-cyclic AMP were without effect on the rate of hexokinase-catalyzed G6P formation. 7. Since even small quantities of G1P or G6P will inhibit hexokinase, it is proposed that phosphoglucomutase and G1P-uridyltransferase play key roles in regulating the flow of exogenous and endogenous precursors into synthesized trehalose.

INTRODUCTION

THE CAPACITYof silkmoths, Hyalophora cecropia, to synthesize trehalose from exogenous glucose has been studied in vivo (Wyatt, 1967; Personal Communication). Larvae incorporated glucose into hemolymph trehalose 300 times faster than did diapause pupae. When studied in vitro, larval and diapause pupal fat body synthesized trehalose from endogenous precursors at rates equivalent to that produced in vitro by larval fat body from exogenous glucose (Jungreis, 1972). While a 70% reduction in fat body glucose phosphorylating capacity occurs between the larval and pupal stages of development (Jungreis, 1975), it can not account for the virtual absence of glucose incorporation by pupal fat body in vitro or in vivo. The regulatory role of hexokinase (E.C. 2.7.1.1.) in carbohydrate metabolism has not been studied in any insect, although a report on the distribution of hexokinase in honey bee body parts contained some kinetic data (Ruiz-Amil, 1962). In this report, the effects on hexokinase activity of sugar phosphates common to the pathways of glycolysis, glycogen and trehalose synthesis were studied in larval and pupal Hyalophora cecropia fat body.

was either homogenized in Tenbroeck glass homogenizers and hexokinase activity measured, or frozen at -20°C prior to analysis. Reagents and chemicals employed were of the highest purity available and were purchased from Sigma Chemical Corporation (St. Louis, Mo). Trehalose-6phosphate (T6P) was a gift from Prof. Stanley Friedman.

Preparation of larval and pupal fat body hexokinase Pupal. Fat body (ca. 40 g), collected from 45 foliage fed pupae (chilled 4 months) was homogenized in 4oral of 100raM Tris-HC1 (pH7.50) [Tris (Hydroxymethyl) Aminomethane] (homogenized pupal fat body) and filtered through two layers (3 cm) of glass wool (pre-rinsed with buffer). Filtered homogenate was then centrifuged at 0°C for 20min at 10,000 g in a SorvaU RC refrigerated centrifuge (Ivan Sorvall, Norwalk, Conn.), the pellet discarded, and the supernatant fraction fractionated at 1°C with 070% ammonium sulfate-specific quantities added slowly over a 20 min period followed by 30 min of additional stirring. Insoluble protein was then removed by centrifugation, and the respective pellets re-suspended in ca. 5.0 ml of 10mM Tris buffer. Resuspensions were dialyzed over a 48 hr period against three changes of 4 litres each of buffer. Fractions of 0-2070 (Fraction 1), 20-35% (Fraction 2), 355070 (Fraction 3), 50-70%o (Fraction 4) and 70+70 ammonium sulfate (Fraction 5) were obtained, with Fraction 4 (purified 13'l-fold) used as a source of enzyme for most of the kinetic studies (Table 1). MATERIALS AND METHODS Larval. Fat body collected either from eight synthetic diet or nine foliage reared feeding fifth instar larvae, and General procedures frozen at -20°C for 7 months, was homogenized in 6 vol Insects, fed wild cherry foliage (Prunus serotina) or syn- (w:v) of 15mM Tris buffer, and centrifuged at 10,000g thetic diet (Riddiford, 1968), were chilled in crushed ice for 20 min. Since soluble or dialyzeable inhibitors or actifor 15 min, after which fat body tissue was dissected free vators of hexokinase were absent under assay conditions, from other adhering tissues. After blotting with glassine the supernatant fractions were decanted and used without paper (Eli Lilly & Co., Indianapolis, Indiana), fat body further purification (Jungreis, 1975). 405

406

ARTHUR M. Table 1 FRACTION

VOLUME (m0

JUNGREIS

Purification of hexokinase SPECIF IC ACTIVITY nanomoles/mg protein-hour

TOTAL PROTEIN (mg)

homogenized pupal fat body

64.0

12.8

12186

filtered through gl~s wool centrifuged at 10,000 x g and dialysed

54.4

21.1

5272

0-20% (NH4)2SO4 (Fraction 1)

5.5

105.7

20-35% (NH4)2SO4 (Fraction 2}

5.0

78,9

35-50% (NH4)2SO4 (Fraction 3)

5A

135.0

50 70% (NH4)2SO4 (Fraction 4)

11.0

70+% (NH4)2SO4 (Fraction 5)

74.4

PUR IF [CATION

0

PERCENT RECOVERED

100

1.6

80.4

109

8.3

7.4

97

6.2

4.9

250

10,5

21.6

167.7

386

13.1

41.4

.67

4120

Assay o1 hexokinase Hexokinase activity was measured by the slightly modified method of Gots & Bessman (1973) as described in Jungreis (1975). Into individual test tubes were pipetted 0.1 ml of either Fraction 3, Fraction 4, or larval homogenate, and 0,5 ml of an assay mixture consisting of (final concentrations): 120 mM Tris-HC1 (pH 7.5), 37.8 mM MgC12, 6-0 mM ATP, and 1-63 mM glucose (containing 0-25 #Ci/tube of D-glucoseJ4C-UL). Tubes were covered with Parafilm and incubated in triplicate at 30°C for 15 or 30 rain. Following incubation, only 2 5% of glucose present in the assay had been converted to glucose-6phosphate (G6P). When the effects of hexose phosphates or other chemicals on the rate of hexokinase-catalyzed G6P formation were studied, final concentrations in the assay were reduced by 1/7th.

Determination of the molecular weight of hexokinase Preliminary determination of the mol. wt of hexokinase was made by Professor Ronald J. Downey, Ohio Unkcr sity, from the calculated sedimentation coefficient using i1~,. method of Martin & Ames (1961). Sucrose gradients wcrc formed using an ISCo Gradient Former and fractionated with an ISCo Density Gradient Fractionater (Instrument Specialties Company, Lincoln, Nebraska). The sedimentation coefficient of hexokinase was determined by centrifuging at 40,000 rev/min 0.2 m of Fraction 4 on 5 ml of a 10/30% linear sucrose gradient (pH 7.8) at 4°C for 20 hr in a SW 50 rotor in a Beckman Model L2-65B ultra-

0.05

1.8

centrifuge equipped with a rotor stabilizer (Beckman Instruments, Inc., Palo Alto, California), The brake was off during deceleration. Samples were automatically run using band forming caps. Yeast alcohol dehydrogenase (3 × recrystallized), a marker protein whose sedimentation coeffi0-72 cient S2o, w of 31 had previously been confirmed on these gradients using other protein markers, was simultaneously run and analyzed spectrophotometrically using the method of Vallee & Hoch (1955). Hexokinase activity in the individual 0.123ml sucrose fractions was determined by the method of Gots & Bessman (1973) following incubation at 40°C for I hr.

Determination of the pH optimum The supernatant from foliage fed larval fat body homogenate was also used as a source of hexokinase. Into individual tubes were pipetted 0.1 ml of supernatant and 0"4 ml of an assay mixture consisting of (final concentrations): 5.2 mM ATP, 31.5 mM MgCIz, 2.0 mM glucose (containing 1.5 ~Ci/tube of D-glucose [~4C]UL) and 180 mM Tris buffer (pH 6.0-9.6). Triplicate assays were incubed for 30 min at 37°C and G6P formed measured by the method of Gots & Bessman (1973). RESULTS

pH optimum and molecular weight of hexokinase He×okinase h a d a b r o a d b a n d of activity between pH's 7-0-8.8 with a n o p t i m u m at p H 8.4, in agreement

3.0

i

i

1,5

."

o e•

o•

• 0

a'''a•a'a°~°e

10%

I

-?..?.7.:...:.<.. I

I

~

L

L

...... . <

•

F r~tio~ Number

Fig. 1. Sedimentation profile for hexokinase fractionated from a linear sucrose gradient (see text). ---, location of marker protein alcohol dehydrogenase.

o~.Io/°\,

Regulation of hexokinase by hexose phosphates

!

i

/

0

,/

.S

•o

I

J

l

l

l

l

l

~

pH

Fig. 2. pH Profile for fat body hexokinase. with values reported for honey bee hexokinase (RuizAmil, 1962) (Fig. 1). At pH's above 9"0 and below 6-8, activity was markedly depressed with no detectable activity at pH's below 6.2. The sedimentation coefficient of Cecropia pupal fat ~o.72 = 13.3, yielding a mol. body hexokinase was ~,zo,w wt of 64,000 (Fig. 2). This value is lower than those reported for yeast (96,600), bovine brain (107,000) and several rat hexokinases (97,000) (Kunitz &

407

McDonald, 1946; Grossbard & Schimke, 1966; Redkar & Kenkare, 1972). K m and V,~ x determinations for larval and pupal

hexokinase Relationships between initial reaction velocity (V) and substrate concentration (S) were determined for larval and pupal fat body hexokinase (Fig. 3), with regression coefficients (slopes), Y and X intercepts determined by least squares fit of the data. The K,, and Vmax toward glucose at constant [ATP] were 1-11 × 10-3 and 1.29 × 10-3M, respectively, for the pupal enzyme, and 1.41 x 10 -3 and 1.75 x 10-3M, respectively, for the larval enzyme. Observed differences in slopes (see Fig. 3) arise from differing activities in the enzyme sources, pupal having 3 x the activity of the larval preparation. These K,,'s are greater than those previously reported for honey bee [4 x 10-SM (Ruiz-Amil, 1962), or rat [Type 1 : 4 . 7 x 10-SM; Type 2 : 2 . 2 x 10-4M; Type 3: 7.0x 10-6M (Grossbard & Schimke, 1966)]. The inhibitor constants (K~) for glucose-l-phosphate (G1P) were virtually indistinguishable in the two enzyme preparations, namely 2.3 + 0-4 x 10-aM calculate~d by the method of Dixon (1953) or 4"6+ 0"3 x 10-3M calculated from the Km in the presence of inhibitor at concentrations of - i [theoretical determination, see Chapter 8 in Dixon & Webb, 1964; (Fig. 4, Table 2)]. These data indicate that larval and pupal fat body hexokinase are probably the same enzyme.

Effects of hexose phosphates on hexokinase catalyzed 91ucose-6-phosphate formation

Irval 7

! .! pupal

I

-4

i

I

I

20 Glueom-1 x 103 MotK

Fig. 3. Lineweaver-Burke plots of reciprocal glucose concentration IS] plotted against reciprocal [V] activity for larval and pupal fat body hexokinase. Larval hexokinase had a K,, and I/maxof 1"41 X 10-3 and 1-75 x 10-3 M, respectively, while those for the pupal enzyme were 1.11 x 10-3 and 1-29 x 10-~M, respectively.

In organisms other than insects, hexokinases are reported to have comparable specificities for glucose and fructose, and like honey bee hexokinase, all are non-competitively inhibited by G6P (Crane, 1963; Crane & Sols, 1953; Rose & O'Connell, 1964; RuizAmil, 1962). The effect of fructose-6-phosphate (F6P) on hexokinase-catalyzed G6P formation has not previously been reported. G6P and F6P are both noncompetitive inhibitors of Cecropia hexokinase with Ki's of 3"0 × 10 - 4 and 4'0 x I0-4M, respectively (method of Dixon, 1953), or 6"0 × 10-4M (theoretical) (Table 2, Figs. 5 and 6), with the Ki's for G6P equivalent to those previously reported for honey bee and rat hexokinase (Grossbard & Schimke, 1966; Ruiz-Amil, 1962; Crane & Sols, 1953). The Ki's for G6P and F6P can be interpreted as evidence of greater affinity by these substrates for hexokinase compared to glucose (K,, = 1'2 x 10 -3 M). However, these hexose phosphates bind to the enzyme at sites other than the active site (Grossbard & Schimke, 1966). Uptake of glucose by larval fat body would be significantly inhibited if G6P and F6P were to accumulate intracellularly. In larval Cecropia, virtually 100~o of the incorporated glucose is recovered as trehalose or glycogen (Jungreis, 1972; In preparation), little if any being oxidized. However, a small though transient increase in the G6P pool size can be inferred from studies on glucose uptake during larval fat body trehalose synthesis (see Fig. 6 in Jungreis & Wyatt, 1972), In mammalian red blood cells, by inference, these glycolytic intermediates fail to accumulate (Minikami et al., 1964; Minikami & Yoshikawa, 1965), although glucose uptake continues to be

408

ARTHUR M. JUNGREIS o

15-

o ,/"'/& o

o /4

/ o

10

•

,/ •

7 .o---°"

"

o..--"

--1S

-i=

-i

-i

i i

-3 Gluco~ 1 Phosphate ImM)

Fig. 4. Dixon plots demonstrating non-competitive inhibition by GIP of hexokinase-catalyzed G6P formation. K~'s of 2.7 x 10 -3 and 1.9 x 10 -3 M, respectively (method of Dixon, 1953), and 4.3 x 10 -3 and 4.9 x 10 -3 M, respectively (theoretical), were determined for pupal and larval fat body hexokinase. Triangles-larval hexokinase: solid-l.63 mM glucose; open-0.81 mM glucose. Circles-pupal hexokinase: solid-0.177 mM glucose; open 0.15 mM glucose.

directly related to the size of the G6P pool (Rose & O'Connell, 1964). The precursor of G6P in the Embden-Meyerhof pathway, G IP, is an obligate intermediate in trehalose synthesis, and though having the same affinity for hexokinase as does glucose, this affinity is only 10% that of G6P's (Figs. 3 & 4, Table 2). While phosphoglucomutase (E.C. 2.7.5.1) catalyzed conversion of G6P to G1P would result in a 10-fold reduction, based on the respective K~'s in hexokinase inhibition, appreciable inhibition would remain unless the intracellular pools of glucose ,> G1P. That this assumption is incorrect was shown by Jungreis & Wyatt Table 2.

Theoretical and calculated Ki's

Concentration of Glucose (raM)

Tissue Fraction 4

(1972) and Wyatt (1967), who were unable to detect measureable quantities of glucose in either larval or pupal fat body. Furthermore, were G6P to be converted to fructose-l, 6-diphosphate (F16P) or uridinediphosphate-glucose (UDPG), inhibition would cease (Figs. 7 & 8). Since glucose is not normally used to any extent by intact fat cells in vitro or in vivo (Jungreis, 1972; In preparation; Jungreis & Wyatt, 1972), rapid removal of G1P, G6P and F6P can arise only by their conversion to neutral substances such as UDPG, trehalose-6-phosphate (T6P) or trehalose. When incubated with physiological levels of these substrates, UDPG, T6P and trehalose were without for

Cecropia fat body hexokinase

it Inhibitor

{mM)

......

theoretical*

1.39

F-6-P

1.18

4.0 x 10 "4

6.2 x 10 -4

0.70

F-9-P

1.25

4.0 x 10.4

5 6 x 10 .4

1.50

G-6-P

1.61

3.0 x 10 .4

7,9 x 10 .4

1.42

G-6-P

1.11

3.0 x 10,4

5.0 x 10 -4

0.19 0.15

G-1-P G-1-P

5.:28 4.33

2,7 x 10 -3 2.7 x 10 .3

4.6 x 10 .3 3.9 x 10 `3

13.6

fifth larval instar fat body, pooled

1.39

G-I-P

from 9 foliage reared animals

0.70

G-I-P

fifth larval instar fat body, pooled

1.39

AIDP

from 8 synthetic diet reared animals

0.70

ADP

1.39

AMP

15.8

--

0.70

AMP

16.0

....

Fraction 3

~ i (M°lar)¢

method of Dixon +

1.9 x 10 .3

7.1 x 10 .3

1.9 x 10 3

2.7 x 10 .3

1.43

....

0.7 x 10 .3

3.29

--

3.95

-

2.2 x 10 .3

-

0.79 x 10 .2 ~ 1.05 x 10 .2

tThe concentration of inhibitor at which a regression line of 1/velociW (ordinate) plotted against concentration of inhibitor intersects the abseisse. +The point on a reciprocal plot of 1/velocity 1/velocity.

X = concentration

versus i

where regression lines

in the form Yi = bXi + Ci have identical Y values and X values.

of inhibitor.

#K i = --i/ (S/Krn +1), where K i = inhibitor constant, S = concentration

of glucose, K m = 1.36 x 10 .3 M.

Y =

Regulation of hexokinase by hexose phosphates

409

12 O

12

9

o

o

o E

8

6 i

0

@

•

3 4

-=

o

-,

l

~

G lucose~6-Phosphate

~

~

,/

s

{raM)

Fig. 5. Dixon plot demonstrating non-competive inhibition by G6P of hexokinase catalyzed G6P formation. Ki's of 6-0 x 10-4M {theoretical) and 3-0 x 10-'*M (method of Dixon 1953) were obtained from these data. Open circles 1.41 mM glucose; solid circles 1.50 mM glucose.

1.5

I

0

1.5

Ecdysone, previously observed to potentiate trehalose synthesis (Jungreis, 1972; In preparation), through an effect on the phosphorylase enzymes (Stevenson & Wyatt, 1964; Wiens & Gilbert, 1967), also causes an increase in intracellular 3. Y-CAMP levels (Applebaum & Gilbert, 1972). The effect of Y5'-cAMP on hexokinase catalyzed G6P formation was therefore investigated. No enhancement of glucose uptake was noted (Fig. 12), indicating an involvement of 3'5'cAMP in trehalose synthesis at a point other than at hexokinase.

Effects of ATP, ADP, AMP and Y5'-cyclic-AMP Honey bee hexokinase was reported to be competitively inhibited by both ATP (Ki = 7.5 × 10-4M) and ADP (K~ = 9.0 × 10-4M), dubious results in light of the absence of Mg 2+ in the assay when hexokinase activity was measured (Ruiz-Amil, 1962). In Cecropia, at concentrations of 5.5-10 × 10-a M, ATP enhanced G6P formation [also in agreement with rat hexokinase (Grossbard & Schimke, 1966)] (see Fig. 8), while ADP and AMP were both un-competitive inhibitors with K~'s of 1.5_+ 1.4 × 10-3M and 0.9 _+ 0"1 × 10 -2 M, respectively (Figs. 10 & 11, Table 2). For rat hexokinases, ADP is a non-competitive inhibitor with Ki's of 2.0-5.0 × 10-aM (Grossbard & Schimke, 1966).

DISCUSSION

Synthesis of trehalose Trehalose, synthesized in insects primarily or exclusively in fat body (Candy & Kilby, 1961; Clegg

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"--.o....... o', "°

-2o

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2 '.5

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41,5

Fig. 6. Dixon plots demonstrating non-competitive inhibition by F6P of hexokinase catalyzed G6P formation. K~'s of 6.0 x 10-4M (theoretical) and 4.0 x 10-4M (method of Dixon, 1953) were obtained from these data. Open circles 1.39 mM glucose; Solid circles 0.70 mM glucose.

effect on hexokinase-catalyzed G6P formation (Figs. 8&9).

+1o

3:0

Fructose-6-Pholphate (raM)

5 ~o

Concentration of Effector

'

' 7.5

'

10

(raM)

Fig. 7. The absence of an effect of FIP, F16P and ATP on hexokinase catalyzed G6P formation. Open box: F1P, 1'39 mM glucose; Shaded box: F1P, 0.70 mM glucose; Open triangle: F16P, 1.39 mM glucose; Solid triangle: FI6P, 0.70 mM glucose; Open circle: ATP, 1-39 mM glucose; Solid circle: ATP, 0.70 mM glucose.

ARTHUR M.

41(1 g

+10 •

t~ ~o

i 1 ~ 1 1

•°

o

•

,

-10

;.1,

l

•

; If:

E

I

o

1.5

3.0

Concentration of Effector (mM)

Fig. 8. The absence of an effect of T6P and UDP-Glucose on hexokinase catalyzed G6P formation. Open circles: T6P. 0.70 mM glucose; Solid circles: T6P. 1.39 mM glucose; Solid box: UDPG. 1.39 mM glucose. & Evans, 1961 ; Murphy & Wyatt, 1965; Wyatt, 19671 is formed by pathways demonstrated in yeast (Cabib & Leloir, 1958), insects (Candy & Kilby, 1961; Murphy & Wyatt, 1965) and helminths (McAlister & Fisher, 1972}:

JUNGREIS

have recently been reported (Jungreis & Wyatt, 1972; Jungreis et al., 1974). However, these changes shed light only on the question of how trehalose steady state levels can be sustained, and add little to questions dealing with the contribution of endogenous and exogenous precursors to synthesized trehalose. Synthesis of trehalose is dependent upon readily accessible pools of G6P and G I P (Steps I & 2); rapid conversion of G1P into UDP-glucose (Step 3); glucosyl transfer (Step 4); the concentration of Mg z+ (which affects Steps 4, 5 and 1); and the rate at which trehalose is removed from the fat cells (affecting Step 4 directly, and others indirectly) (Friedman, 1971; Jungreis et al., 1974; Jungreis & Wyatt, 1972; Murphy & Wyatt, [965; McAlister & Fisher, 1972). Both G6P and G IP inhibit hexokinase catalyzed G6P formation. Formation of G6P stimulates Step 2c (Martensen el a/., 1973}, while conversion of G6P to G l P (Step 2a), an intermediate in both glycogen and trehalose synthesis, effectively reduces both phosphorylase b activity (Step 2b) and enhances the comparative rate at which glucose is phosphorylated by reducing noncompetitive allosteric inhibition by G6P at Step la

MgATP + Glucose---* MgADP + Glucose-6-phosphate (Hexokinasc, E.C. 2.7.1.11 MgATP + Fructose--* MgADP + Fructose-6-phosphate (Hexokinase, E,C. 2.7.1.11 Fructose-6-phosphate ~ Glucose-6-phosphate (Phosphoglucose isomerase, E.C. 5.3.1.9i Glucose-6-phosphate,-~ Glucose-l-phosphate (Phosphoglucose mutase, E.C, 2.7.5.1) Glycogen° + Ortho-phosphate-+ Glucose-l-phosphate + Glycogen.i-~ (Phosphorylase h or Phosphorylase a. E.C. 2.4.1.1) Step 2c. Pbosphorylase a -~-~Phosphorylase b Phosphorylase phosphatase, E.C. 3,1.3.17 & Phosphorylase kinase, E.C. 2.7.1.38) Step. 3. Glucose-l-phosphate + Uridyl-triphosphate--~Pyrophosphate + Uridyldiphosphate-g~ucose iGlucose-l-phosphate uridylyltransferase, E.C. 2.7.7.9) Step 4a. Uridyldiphosphate-glucose+ Glucose-6-phosphate--* Uridyldiphosphate + Trehalose-6-Phosphate (Trehalose6-phosphate synthetase, E.C. 2,4.1.15) Step 4b, Uridyldiphosphate-glucose + Glycogen,---,Uridyldiphosphate+ Glycogcn,~, ~ !Glycogen synthetase, E.C 2.4.1.11) Step 5. Trehalose-6-phosphate + HOH--~ Trehalosc + Orthophosphate (Trehalose-6-phosphatasc, E:C. 3.1.3.12)

Step Steb Step Step Step

I a. lb. lc. 2a. 2b.

Trehalose-6-phosphate synthetase (Step 4a) has been shown to be "the rate limiting enzyme" in the synthesis of trehalose, being both stimulated by Mg 2+ and inhibited by trehalose (Murphy & Wyatt, 1965; McAlister & Fisher, 1972). Changes in the concentrations of trehalose and Mg 2+ in fat cells, which can account for developmental differences in steady state levels of trehalose in hemolymph and fat body,

/

-=

o o

oJ

.y

2 +10 2

o~

v

g "6 "=~ 0

,

E [~:

O~

~-O

u-

.E

g g

I

-10

3.0

.E

o

0

1()

I

20 Trehalose

30

1

40

(mM)

Fig. 9. The absence of an effect of trehalose on hexokinase catalyzed G6P formation. Solid circles: 1.39 mM glucose; Open circles: 0.70 mM glucose.

1.5 1.5 0 Adenosine 5' di Phosphate (raM}

i

3.0

Fig. 10. Dixon plots demonstrating un-competitive inhibition by ADP of hexokinase catalyzed G6P formation, A K~ of 1"4 × 10 3 M was obtained by the method ot" Dixon (1953). Solid circles: 1.39 mM glucose: Open circles: 0.70 mM glucose.

Regulation of hexokinase by hexose phosphates

411

"7,

o

=

-15

-10

-S 0 Adenosine5' Monopho~ohate (mMI

5

'

10

Fig. 11. Dixon plots demonstrating un-competitive inhibition by AMP of hexokinase catalyzed G6P formation. A K of 0'9 x 10-z M was obtained by the method of Dixon (1953). Open circles: 1.39 mM glucose; Solid circles: 0"70 mM glucose. (Grossbard & Schimke, 1966). Thus, the roles of UDP-glucose, T6P and trehalose in controlling Step 1 are of considerable importance in understanding the in vivo regulation of trehalose synthesis. Conversion of G1P to UDP-glucose removes the remaining inhibition at Step la resulting from glucose uptake (see Fig. 8), therein promoting Steps 3 and 4. The Km's for UDP-glucose in Steps 4a & 4b are 3-0 x 10 -4 and 1.6 x 10 -a M, respectively (Murphy & Wyatt, 1965). Thus, in the presence of G6P, UDPglucose will be incorporated preferentially into trehalose over glycogen. The following models are proposed to explain how the flow of glucose derived from exogenous and endogenous sources is controlled during the in vivo synthesis of trehalose. Regulation of trehalose synthesis in Hyalophora cecropia pupal fat body The quantity and activity of hexokinase at Step la will determine the extent to which exogenous glucose is incorporated into trehalose, although this is also affected by Steps 2a and 3. The activity of hexokinase in pupal fat cells is only 10~ of that in larval cells

#.o

+10 O

o" O,

•

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-10

I I

V O

g t-

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x

J

N

i

x

I

x

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~

3':5'-cyclic AMP (Molar)

Fig. 12. The absence of an effect of 3':5'-cyclic AMP on hexokinase catalyzed G6P formation. Open circles: 1.39 mM glucose; Solid circles, 0-70 mM glucose. J~ ,,(u) 5 V 3 B

,

(Jungreis, 1975). Pupal fat cells must then rely primarily upon endogenous precursors such as glycogen for the synthesis qf trehalose (Jungreis, 1972; In preparation). Based upon the proportion of phosphorylase in the a (active) form (Stevenson & Wyatt, 1964; Wiens & Gilbert, 1967), glycogen will be degraded more slowly in diapause pupal than larval fat body, with the rate of G1P incorporation into trehalose directly proportional to the pool of UDP-glucose (see Murphy & Wyatt, 1965). However, glycogen breakdown is also dependent, although only indirectly, upon Step 2a, with the conversion of G1P to G6P stimulating phosphorylase b activity and Step 2c (Martensen et al., 1973). The K m for UDP-glucose in Step 4a is significantly less than for Step 4b (Murphy & Wyatt, 1965), and UDP-glucose will be channelled into trehalose as long as newly syntllcsizcd trchalose is removed from the intracellular milieu at a rate equal to synthesis. When trehalose inhibition at Step 4a becomes pronounced, synthesis ceases and UDP-glucose will be re-incorporated into glycogen (Murphy & Wyatt, 1965; McAlister & Fisher, 1972). Following injury or during incubation in Jungreis & Wyatt (1972) modified Reddy & Wyatt's medium (1967), release of trehalose is promoted (Jungreis, 1972; Jungreis & Wyatt, 1972). This response occurs because intracellular trehalose, egressing slowly from fat cells in vivo, rapidly egresses in vitro (Jungreis & Wyatt, 1972), thereby reducing trehalose inhibition at Step 4a and promoting the flow of UDP-glucose into trehalose. The proportion of phosphorylase in the a form also increases following injury, promoting the formation of both UDP-glucose and G6P (Stevenson & Wyatt, 1964; Wiens & Gilbert, 1967; S. Wyatt, unpublished; Ziegler & Wyatt, personal communication). If the proportion of phosphorylase in the a form were initially high, then G6P derived from Step 2a would promote Step 2c (Martensen et al., 1973), and the rate of trehalose synthesis would decrease. However, preferential movement of G1P into UDPglucose vs G6P will retard Step 2c and promote Step 4a. The enhanced rate of trehalose synthesis will continue under these experimental conditions as long as the relative UDP glucose pool is large at the expense of the G6P pool. When trehalose inhibits Step 4a, the pool size of G6P will be increased, stimulating Steps 2c and 4b. Also, the flee energy change of phosphoglucomutase, calculated from in vivo levels of G1P

412

ARTHUR M. JUNGREIS

and G6P favors the formation of G6P (Lehninger, 1970). Therefore, regulation at Step 2a is important in reducing both the rate of trehalose synthesis from glycogen precursors (Step 2c), and in inhibiting glucose uptake at Step la. Regulation of trehalose synthesis in Hyalophora cecropia larval Jat body Larval fat body differs from that of pupae in many respects. (1) Trehalose readily diffuses into and out of larval but not pupal fat cells (Jungreis & Wyatt, 1972). (2) The concentration of Mg 2+ in larval cells (20 mM) is double that of pupal fat cells (10 raM) (Jungreis et al., 1974), which affects Step 4a at the expense of 4b. (3) The proportion of phosphorylase in the a form is greater (Stevenson & Wyatt, 1964; Wiens & Gilbert, 1967). (4) The capacity to phosphorylate glucose (Step la) is significantly greater (Jungreis, 1975). (5) The capacity of partially purified fat body extracts to synthesize glycogen is significantly reduced in larval relative to pupal fat body (Murphy & Wyatt, 1965). In the absence of exogenous glucose, and assuming the presence of adequate glycogen stores, regulation of trehalose synthesis will be by the same mechanisms utilized by pupal fat cells incubated in vitro. However, in the presence of exogenous glucose, regulation will differ greatly. Hexokinase activity will control Step la, but the formation of G1P will be controlled by Step 2a (which favors G6P formation) and Step la. Both glucose and G6P promote Step 2c (Martensen et al., 1973), thereby reducing the rate of glycogen degradation and G1P formation via Step 2b. The conversion of G6P to G1P will promote Step 3, which in turn promotes Step 4a. With UDP-glucose no longer exerting an inhibitory effect on Step la (Fig. 8), formation of G6P will again be promoted, although G6P so formed is immediately removed via Step 4. UDP-glucose has a greater affinity for T6P-synthetase than for glycogen synthetase (Murphy & Wyatt, 1965). UDP-glucose will therefore combine preferentially with G6P to form T6P (vs glycogen,--, glycogen, + ,), which is in turn hydrolysed to form trehalose. Since phosphorylase b requires the presence of G6P, and assuming the presence of only a small pool of G6P, the rate of glycogen degradation will be proportional to phosphorylase present in the a form. Were trehalose to remain in the fat cells, it would directly inhibit Step 4a, indirectly inhibit Steps l a, 2a and 3, and would promote the flow of UDP-glucose into glycogen (Step 4b). If steps 2a and 3 were not rate limiting, except in the case where glycogen synthesis was also blocked (a condition leading to control at Step l a), glucose uptake would be constant whether or not trehalose was being synthesized. [This latter condition (inhibition of both trehalose and glycogen synthesis) is not observed in fat body in vitro, since the presence of 100 mM trehalose in the incubation medium failed to appreciably inhibit trehalose or glycogen synthesis from exogenous glucose (Jungreis, 1972; In preparation).] Since this state has not been observed, it is proposed that controls at Step 2a (Phosphoglucose mutase) and Step 3 (G1P-uridylyl transferase) are responsible for controlling the flow of precursors into trehalose.

Role of horlTu)nes m the regulation of H yalophora cecropia .tat body trehalose synthesis

The role played by hormones in the "'closed system" approach to trehalose synthesis t a k e n in the preceding sections of the discussion must also be considered. No effects of corpora cardiaca extracts on H. cecropia trehalose synthesis have been observed (see Wyatt, 1972, for a discussion of hyperglycemic hormones), in going from the larval to the diapause pupal stages of development, the titer of ecdysone in hemolymph is markedly depressed, being restored only with the initiation of pharate adult development (see Truman et al., t974). Ecdysone has been shown to affect H. cecropia carbohydrate metabolism, both by changing fat cell penetrability to and release of sugars (Jungreis & Wyatt. 1972), and by influencing the proportion of fat body phosphorylase present in the b or a forms (Stevenson & Wyatt, 1964; Wiens & Gilbert, 1967). While the effects of ecdysone in mediating early pharate adult development have been shown to be mediated by an increase in intracellular 3'5'-cyclic AMP levels (Applebaum & Gilbert, 1972), the titer of ecdysone in vivo appears to play at most only a small role in controlling the flow of precursors into trehalose in vitro. Were ecdysone directly involved, the proportion of precursors incorporated by larval fat body into trehalose would shift with time of incubation from exclusively exogenous (high ecdysone titer characteristic of larvae) to exclusively endogenous (low ecdysone titer characteristic of pupae), events never observed. Larval fat body converts virtually 100% of exogenous glucose (and no endogenous precursors) into trehalose or glycogen. It therefore seems reasonable to view the incorporation of exogenous glucose into G6P, and the formation of G I P and G6P from glycogen to be two aspects of a self-regulating "closed system". That cyclic nucleotides or hormones influence carbohydrate metabolism in If. cecropia is undisputed (see Jungreis, 1972). Similarly, the possibility that changes in intracellular cyclic nucleotide levels result from rather than cause a shift in the flow of substrates into synthesized trehalose has yet to be excluded. Acknowledgenwnts Supported m part by Grants from the National Science Foundation (GB-43457) and Ohio University Research Council (No. 41(IL

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