Diabetologia 9, 299--302 (1973) 9 by Springer-Verlag 1973

Time Course Studies of Glucose-Induced Changes in Glucose-6-Phosphate and Fructose-l,6-Diphosphate Content of Mouse and Rat Pancreatic Islets S. J. H. Ashcroft, K. Capito a n d C.J. Hedeskov Department of Bioehenaistry A, University of Copenhagen, Denmark l%eeeived : F e b r u a r y 9, 1973, accepted 5lay 4, 1973

Summary. The concentrations of glucose 6-phosphate (G6P) and frnctose 1-6-diphosphate plus triose-phosphates (FDP + TPs) were measured in isolated islets of Langerhans from mice and rats after a sudden increase in extracellular glucose coneentration from 0.5 to 3.4 mg/ml. I n mouse islets, the contents of G6P and (FDP - - T P s ) were both ~aised after a two minute incubation at the high glucose concentration and remained elevated for at least 30 main. I n rat islets, the G6P bat not the (FDP-i-TPs) content was increased after a 5 rain exposure to high glucose. After a 30 rain incubation, both G6P and ( F D P " TPs) contents were higher t h a n at low glucose coneentration. The (G6P)/(FDP + TPs) ratio was some tenfold

higher in mouse islets than in rat islets. Increasing extracellular glucose concentration was associated with an increase in the (G6P)/(FDP + TPs) ratio. The results are consistent, with the increased glyeolytie rate in response to a raised extraeellular glucose concentration arising primarily from an increase in the rate of phosphotwlation of glucose, and with the hypothesis that the insulin secretory response to glucose m a y be mediated b y a metabolite of the sugar.

Introduction

Materials and Methods

To s u b s t a n t i a t e the hypothesis t h a t mctabolites of glucose or conditions associated with glucose m e t a b olism, r a t h e r t h a n glucose itself, trigger i n s u l i n release from the islets of L a n g e r h a n s [1, 2], it would be necessary to show t h a t changes i n c o n c e n t r a t i o n s of metabolites or cofactors occur at least as fast as i n s u l i n secretion s~dtches on after a glucose pulse. In in rive experiments with islets from obese hyperglycemic mice a 400% rise i n G6P 1 c o n c e n t r a t i o n w i t h i n 90 sec has been d e m o n s t r a t e d [3]. However, later M a t s c h i n s k y et al. [4] again using a n in vivo approach - - found no changes i n the c o n c e n t r a t i o n s of G6P, 6PG 1, A T P or phosphocreatine i n r a t islets w i t h i n 1 - - 5 m i n after a glucose pulse. W e felt t h a t this species difference i n metabolic response possibly could be explained b y differences i n anaesthesia, in the degree of h y p o x i a a n d stress and/or other e x p e r i m e n t a l conditions difficult to control in vivo a n d have therefore i n in vitro e x p e r i m e n t s measured the c o n c e n t r a t i o n s of G6P a n d ( F D P + TPs) 1 i n isolated islets of L a n g c r h a n s from b o t h mice a n d rats after a s u d d e n rise in extracellu]ar glucose concentrat i o n from 0.5 to 3.4 mg/ml. Significant a n d fast changes in the c o n c e n t r a t i o n of metabolites were d e m o n s t r a t e d i n both rat- a n d mouse islets although most p r o n o u n c e d i n mouse islets.

Purified luciferase from Achromobacter fiseherii and FMN were from Signaa Chemical Co. Myristic aldehyde was from Aldrich Chemical Co. Other enzymes and coenzynaes used were from Boehringer. t t u m a n albumin was from Behringwerke AG. All other reagents were of analytical grade. All glassware used was cleaned in 1.5 N nitric acid and washed in double distilled water.

1 Abbreviations used: G6P: glucose-6-phosphate, F D P + TPs: fructose-l,6-diphosphate plus triose phosphates, 6PG: 6-phosphogluconate.

Key words: Islet metabolism, insulin release, hexose phosphate, luciferase assay of pyridine mmleotides.

Preparation of Islets Islets were isolated b y a collagenase method [5] from the pancreas of male Wistar rats weighing 150-- 175 g or of male T. O. mice weighing 22--26 g. All animals were starved overnight.

Incubation of Islets Batches of 7--10 islets (for glucose-6-phosphate assay) or 20--40 islets (for fructose-l,6-diphosphate assay) were preincubated for 30 m/n at 37o in stoppered glasstubes with 21 ~1 gassed (O2:CO2 95:5) Krebs bicarbonate buffer [61] eontaiuing 0.5 nag glucose/ml and 1 mg h u m a n albumin/ml. After preincubation, the glucose concentration of the medium was raised b y injecting 7 ,ul Krebs bicarbonate buffer containing 12 nag glucose/ml and 1 nag h u m a n albumin/nal. After the time indicated in the tables the metabolism was stopped b y injecting 28 ~1 0.1 N HC1 (for G6P assay) or 1 N I-IC1 (for F D P assay). The low glucose controls were prepared by adding the I:[C1 iramediately after the preincubation and then later the 7 Krebs buffer with high glucose content. Blank values were obtained by incubating medium without islets. Before assaying for G6P or F D P all tubes were sonicated for 20 see at position 1 on a Branson Sonifier B-12. F D P and G6P standards were made up in a Krebs bicarbonate medium-HC1 mixture similar in composition to the acidified incubation medium in order to account for

300

S . J . I-L A s h c r o f t et al. : G6P and F D P Content, of Mouse and I~at P a n c r e a t i c Islets

any interference b y t h e m e d i u m reagents in t h e m e t a b o l i t e assays.

Glucose.6.Phosphate Assay 40 ~xl of t h e sonicate or s t a n d a r d G6P solutions were analysed for G6P b y adding 27 Ezl of 0.2 M trisbuffer p i t 8.0, 1.5 al 1 m M N A D P + and 1.5 41 glucose-6-phosphate d e h y d r o g e n a s e (0.35 U/ml). A f t e r ' i n c u b a t i o n for 60 m i n at room temperature the content of NADPH was measured in triplicate by the luciferase assay described below.

_Fructose-,1-6Diphosphate Assay 40 41 of the sonieate or s t a n d a r d F D P solutions were analysed for F D P and triose p h o s p h a t e s b y adding 27 txl 0.4 M triabuffer containing 0.5 N K O t t , 1 m M m e r e a p t o :

Diabetologia

s a t u r a t e d solution of m y r i s t i c a l d e h y d e in e t h a n o l and 20 ~l of t h e sample to be d e t e r m i n e d were a d d e d to t h e tubes. The tubes were allowed to stand at r o o m t e m p e r a ture in t h e dark for 10-- 15 m i n a n d t h e assay was s t a r t e d b y adding 40 ~1 of a luciferase-solution containing 1 m g lueiferase/ml p h o s p h a t e buffer p H 7.0 to t h e tube. The t u b e was q u i c k l y r e m o v e d f r o m t h e dark, m i x e d on a V o r t e x - G e n i e and transferred to a c o n v e n t i o n a l glass scintillation vial placed in t h e liquid scintillation spectrom e t e r a n d c o u n t e d for 0.I min. The t i m e passing (15 see) f r o m t h e a d d i n g of t h e luciferase u n t i l t h e s t a r t of t h e counting was a c c u r a t e l y standardized b y always adding t h e lueiferase w h e n t h e p r i n t i n g out of t h e results of t h e preceeding t u b e started. A P a c k a r d TriCarb scintillation spectrometer, m o d e l 2002

Table 1. Glucose-6-phosphate and fructose-I, 6-diphvsphate in islets of Langerhans

Mouse Islets Control (glucose 0.5 mg/ml) G 6 P (pmol/10 islets) o/o a b o v e control F D P -- T P s (pmol/10 islets) % a b o v e control G6P F ] ) P --~ T P s ratio

Duration of incubation with glucose 3.4 mg/ml 2 rain

5 rain

30 min

5.0 -j= 0.9

(11)

12.4 • 0.5 b 148

(3)

12.1 :!- 1.0 a 142

(12)

15.5 :~: 1.5 a 210

(17)

0.37 if= 0.05

(26)

0.71 ___'0.09 c 92

(5)

0.72:~ 0.12 c 95

(32)

0 . 6 3 • 0.05 a 70

(30)

13.5

17.5

16.8

24.6

Rat Islets G6P (pmol/10 islets) ~ a b o v e control F D P ~- T P s (pmol/10 islets) ~ a b o v e control G6P ratio F D P + TPs

2.5 • 0.2

(28)

4.2 • 0.3 a 68

(26)

5.5 =~: 0.3 a 120

(30)

1 . 3 2 ~ 0.12

(33)

1.59 :[: 0.15 a 20

(31)

2.06--' 0.20 b 56

(32)

1.9

2.6

2.7

a Different f r o m control w i t h p < 0 . 0 0 1 b Different f r o m control w i t h p < 0 . 0 0 5 c Different f r o m control w i t h p < 0.02 a NS, p > 0 . 2 Batches of mouse or r a t islets were p r e i n c u b s t e d for 30 m i n in 21 ~1 bicox'bonate m e d i u m containing a l b u m i n (1 mg/ml) and glucose (0.5 mg/ml). The m e d i u m glucose c o n c e n t r a t i o n was t h e n raised to 3.4 m g / m l b y injection of 7 ~zl m e d i u m containing 12 m g / m l of glucose. A f t e r t h e t i m e s indicated islet m e t a b o l i s m was stopped b y t h e injection of HC1 and t h e islet c o n t e n t s of G6P and ( F D P -~ TPs) were m e a s u r e d as described in t h e text. The d a t a are expressed as m e a n • S.E.M. w i t h t h e n u m b e r of batches of islets u n d e r each c o n d i t i o n g i v e n in p a r e n t h e ~ s . e t h a n o l and 1 m M NaIIpAsO 4, 1.5 41 1 m M N A D + and 1.5 ~1 of an e n z y m e m i x t u r e eontair[ing triosephosphateisomerase (120 U/ml), g l y e e r a l d e h y d e - 3 - p h o s p h a t e dehydrogenase (40 U / m l ) and Mdolase (4.5 U / m l ) . A f t e r i n c u b a t i o n for 90 m i n at r o o m t e m p e r a t u r e the c o n t e n t of NADI-I was m e a s u r e d in duplicate b y t h e lueiferase assay described below. The assay measures the sims of f r u c t o s e - l , 6 - d i p h o s p h a t e and triose p h o s p h a t e s ( F D P -~ TPs).

Luciferase Assay D e t e r m i n a t i o n of NAD]-I and NADPI-I was based on t h e m e t h o d described b y P . E . S t a n l e y [7]. Counting p r o c e d u r e : P o l y p r o p y l e n e tubes (1.3 • 5.0 era) were soaked o v e r n i g h t in w a t e r and w a s h e d w i t h double distilled water. 1 m l of a 0.1 M trisbuffer pI-I 7.3 containing 1 m M m e r c a p t o e t h a n o l was m i x e d w i t h 2 ~1 F M N (0.1 m g F M N / m l 0.1 M p o t a s s i u m p h o s p h a t e blfffer pl-I 7.0) and placed in a p o l y p r o p y l e n e tube. 10 ~1 of a

o p e r a t e d at r o o m t e m p e r a t u r e was used. The photomultipliers were switched out-of-coincidence. Amplificat i o n was 100~ and t h e discriminators were set b e t w e e n 10 and infinity.

Results Since islet G 6 P a n d F D P w e r e d e t e r m i n e d f r o m standard curves prepared using authentic G6P and F D P t a k e n t h r o u g h t h e s a m e p r o c e d u r e s , no correct i o n s for r e c o v e r y w e r e n e e d e d . T y p i c a l s t a n d a r d c u r v e s for G 6 P a n d F D P in t h e l u e i f e r a s e a s s a y are s h o w n in t h e F i g u r e . T h e c o n c e n t r a t i o n s of G 6 P a n d ( F D P @ T P s ) in m o u s e a n d r a t islets a t v a r i o u s t i m e s a f t e r raising t h e

S.J.I-I. Ashcroft et al. : G6P and FDP Content of Mouse and Rat Pancreatic Islets

Vol. 9, No. l, 1973

extraeellular glucose concentration from 0.5 to 3.4 mg/ml are given in the Table. I n mouse islets, the contents of G6P and (FDP + TPs) were both significantly raised after a two minute incubation at the high glucose concentration and remained elevated for at least 30 rain. I n rat islets, the G6P but not the ( F D P - ~ T P s ) content was significantly increased after a 5 rain exposure to high glucose (the shortest time interval tested). After a 30 min incubation both G6P and ( F D P @ T P s ) were significantly higher than at low glucose concentrations.

E o 70

X6a s0

m 4c s 3c

g 2c

P

I

1 picomoles

I

I

I

2 of

G6P or

I

3

I

I

4

FDP

Fig. 1. Standard curves for G6P and FDP using the luciferase assay. NADPH was generated from G6P using NADP + and glucose-6-phosphate dehydrogenase. NADIt was generated from FDP using NAD +, aldolase, triose phosphate isomerase and triose phosphate dehydrogenase. The reduced pyridine nucleotides formed were then determined by the luminescence produced by bacterial luciferase using a scintillation counter as detector. See the text for experimental details. Each point plotted is the mean of six determinations. @--@ FDP, A - - A G6P Although mouse and rat islets are of similar size, there were differences between the absolute and relative contents of G6P and (FDP + TPs) in the two species. The G6P content was higher in mouse islets than in rat islets whereas the reverse was true for the (FDP + TPs) content. As a result the [G6P]/[FDP @ TPs] ratio was some tenfold higher in mouse islets than in rat islets. In mouse islets, increasing extracellular glucose coneentration was associated with an increase (approx. 100%) in the [ G 6 P ] / [ F D P + T P s ] ratio; in rat islets this increase was much less marked.

Discussion

The view that glucose must be metabolized in order to elicit insulin release envisages that effects on insulin release of changes in the extracellular glucose concentration are mediated, at least in part, by changes in the intracellular concentration of one or more metabolites

30 t

formed from glucose in the fi-eell. I t should be emphasized that neither the magnitude nor the direction of the changes in such a hypothetical trigger metabolite need parallel the changes in insulin secretion rate ; however, the rapidity of the metabolite concentration change should be comparable to that of the secretory response. I n islets from several species, increasing extracellular glucose concentrations have been shown to give rise to increased islet content of hexose phosphates [3, 8, 9]. In an earlier study [8], we showed that a correlation existed between insulin release rate and hexose phosphate content of mouse islets under various conditions. Glucose-6-phosphate was shown to attain a steady state level in mouse islets after 15 rain of incubation but the rapidity of changes in the G6P level in response to changes in extracellular glucose concentration was not further investigated. Earlier studies of the kinetics of changes in islet content of glycolytie intermediates have yielded conflicting results; whereas very rapid ( < 90 sec) changes were observed in islets from obese-hyperglycemic mice on rapid elevation of blood glucose in vivo [3], no changes were found in rat islets after 5 rain [4]; 60 min after elevation of blood glucose rat islets did show an increase in hexose phosphate content. When similar measurements were carried out on islets from a perfused rat pancreas preparation, no significant changes in islet metabolic intermediates were seen at any time up to 40 min after acute elevation of the extraeellular glucose concentration [4]. I t was argued that the rapid initial insulin secretory response to sugars could not be mediated by these intermediates. A recent report, however, has described fast (1 rain) decreases in the islet content of glyeolytic intermediates in islets from duct-ligated rat pancreas in response to high glucose [t0]. Our data show that mouse and rat islets both evince significant and rapid changes in G6P content .after elevation of extrace]lular glucose concentration. (FDP @ TPs) content is also increased after 30 rain; however, whereas the change in (FDP @ TPs) content occurs in less than 2 rain in mouse islets the rate of increase of (FDP + TPs) concentration in rat islets is sluggish, no significant increase being seen after 5 min. These results confirm that there are indeed differences in the metabolic responses to an increased extracellular glucose concentration between mouse and rat islets but suggest that these differences are quantitative rather than qualitative. I n particular they indicate that changes in hexose phosphate content of rat islets although less marked than for mouse islets occur within 5 min after raising the glucose concentration. These findings are thus consistent with the hypothesis that the insulin secretory response to glucose may be mediated by a metabolite of the sugar. The relative amounts of G6P and (FDP @ TPs) in isolated rat islets in the present study are similar to those reported for islets in a perfused rat pancreas preparation [4]. I t is striking that in mouse islets the

302

S.J.H. Ashcroft et at. : G6P and FDP Content of Mouse and R~at Pancreatic Islets

[G6PJ/[FDP-~-TPs] ratio is some ten-foid higher than in the rat islets. The enzymic basis for this difference is not known. I n mouse islets the high Km for glucose utilization [11] is to some extent explicable in terms of the high Km glueose-phosphorylating activity and the glucose-inhibited glucose-6-phosphatase activity [3, 12]; in view of the failure to demonstrate these activities in rat islets [4, 13] and the low Km for glucose of hexokinase, the increased glucose oxidation rate in rat islets as the glucose concentration is raised from 3 to 20 ml~I [14] is puzzling. I t has been suggested that the phosphofruetokinase reaction may be the step that is stimulated by the addition of glucose; the evidence for this was a decrease in the [G6P]/[FDP ~ TPs] ratio in rat islets when the extracellular glucose concentration was raised from zero to 20 mM [4]. Under the conditions of our experiments, however, no such decrease was observed; indeed in mouse islets a marked increase was found. These data do not support the view that phosphofructokinase was activated other than by a rise in substrate concentration. They are consistent with the increased glycolytie rate in response to a raised extraeellular glucose concentration arisb~g primarily from an increase in the velocity of the ratedetermining step for glucose uptake, which in view of the rapid permeation of glucose into islets [3, 15], is likely to be phosphorytation of glucose. Aclcnowledgsments. This work was supported by grants

from the Danish Medical lgesearch Council (grant 512-1512), the P. Carl Petersen Foundation and Nordisk Insulinfond. We thank Anne-Marie Fabricius and Anette Garval for expert and enthusiastic technical assistance.

References 1. Randle, P.J., Asheroft, S.J.H., Gill, J.R. : In: Carbohydrate Metabolism and its Disorders, p. 427. Ed. Dickens, F., I:~andle, P.J., Whe]an, W.J. London: Academic Press 1968. 2. Grodsky, G.M., Butts, A.A., Bennet, L.L., Vcella, C., Mc Williams, N. B., Smith, D. F. : Amer. J. Physiol. 205, 63 (1963). 3. Matsehinsky, F. M., Ellerman, J. E. : J. biol. Chem. 243, 2730 (1968). 4. Matschinsky, ~LM., Landgraf, lg., Ellerman, J.E., It:otler-Bra.jtburg, J.: Diabetes 21 (Suppl. 2) 555 (t972). 5. Coll-C~rcia, E., Gill, J.I~. : DiabetoIogia 5, 61 (1969). 6. Krebs, I-LA., Henseleit, :K. : Hoppe-Seylers Z. physiol. Chem. 210, 33 (1932). 7. Stanley, P.E. : A~alyt. Biochem. 39, 441 (1971). 8. Ashcroft, S.J.H., I-Iedeskov, C.J., t~andle, P.J.: Biochem. J. 118, 143 (1970). 9. Montague, W., Taylor, K.W.: Bioehem. J. 115, 257 (1969). 10. Mayhew, D.A., Corkey, B.E." Diabetes 21 (Suppl. 1), 61 (1972). 11. Ashcroft, S. J. H., Weerasinghe, L. C. C., Randle, P. J. : Biochem. J. 126, 525 (1972). 12. Ashcroft, S.J.H., l~andle, P.J.: Biochem. J. 119, 5 (1970). 13. T~ljedal, I.-I3.: Biochem. J. 114, 387 0969). 14. Jarrett, l~.J., Keen, H.: Lancet, 1966 I, 633. 15. Hellman, B., Sehlin, J., T~ljedal, I.-B.: Biochem. biophys. Acts, 241, 147 (1971). Dr. C.J. I-Iedeskov Universitetets biokemiske Institut A Juliane Mariesvej 30 DK-2100 Copenhagen Denmark