The Effects of Glucose, N-Acetylglucosamine, Glyceraldehyde and Other Sugars on Insulin Release in Vivo S

Diabetologia 11,279-284 (1975) by Springer-Verlag 1975

The Effects of Glucose, N-Acetylglucosamine, Glyceraldehyde and Other Sugars on Insulin Release in Vivo S. J.H. Ashcroft and J.R. Crossley

Department of Biochemistry, University of Bristol, England

Received: February 18, 1975, and in revised form: April 28, 1975

Summary. The specificity for carbohydrates of insulin secretory duced hyperglycaemia, but peak insulin concentrations occurred responses in vivo was studied. Test sugars were injected via a left before any change in plasma glucose concentration. No evidence femoral vein cannula into conscious rats. Blood samples collected was obtained for a stimulatory effect of galactose on insulinrelease. over the ensuing 60 min via a left femoral arterial cannula were Infusion for 60 rain of N-acetylglucosarnine produced a sustained assayed for plasma insulin and glucose, and, in some experiments, elevated plasma insulin concentration and significant hypogly- for N-acetyl glucosamine. Whereas L-glucose or saline produced no caemia. The present in vivo results agree with previous in vitro significant changes in plasma insulin or glucose concentrations, observations, and could indicate a role for sugars other than glucose D-glucose, N-acetylglucosamine, D-glucosamine, fructose, D-glyc- in the regulation of insulin release. eraldehyde and DL-glyceraldehyde were potent secretagogues. Simultaneous injection of mannoheptulose abolished the insulin- Key words: N-acetylglucosamine, insulin release, glyceraldehy- otropic action of glucose and N-acetylglucosamine, but not of DL- de, glucose, glucosamine, fructose, galactose, mannoheptulose, glu- glyceraldehyde. Fructose, glucosamine, and DL-glyceraldehyde in- coreceptor.

Detailed studies of the specificity of the insulin se- been conclusively established. Strong evidence for the cretory response to sugars have been performed with involvement of a metabolite has been provided by the mouse and rat islets of Langerhans in vitro [1, 2]. Of a close correlations observed between the metabolic large number of sugars tested only glucose, mannose, activities of exogeneous substrates and their ability to glyceraldehyde and, to a small extent, glucosamine initiate insulin release in isolated mouse islets [1, 2]. In have been shown to initiate insulin release when test- addition the specificity of the inhibitory action of ed singly at a concentration of 20 mM in the presen- mannoheptulose is that which would be predicted for ce of caffeine. A second group of sugars were ineffec- a metabolic basis for initiation: i.e. mannoheptulose tive alone, but in the presence of an initiator such as inhibits the metabolism of glucose but not of glyceral- glucose or D-glyceraldehyde elicited a marked increa- dehyde, and blocks glucose - stimulated but not gly- se in insulin release rate: these potentiators of release ceraldehyde-stimulated release [2, 3]. were fructose [1, 2, 4], N-acetylglucosamine [1, 2], L-glyceraldehyde [3] and (in the rat islets only) galac- Much less information is available on the specificity tose [1]. These findings were interpreted in terms of a in vivo of the insulin secretory response to sugars. two-site glucoreceptor model [2]. On this model the Results of previous in vivo studies have been reviewed glucoreceptor is envisaged to possess two different [5]. However early investigations (e. g. 6) did not have classes of sub-units: an initiator unit sensitive to gluco- the benefit of the sensitive and specific insulin radio- se, mannose, glyceraldehyde and glucosamine (or a immunoassay, and later studies (e. g. 7) have been metabolite thereof), and a potentiator unit of broader confined to a more restricted range of sugars. We specificity. Binding at the potentiator site only elicits report below the results of an investigation to establish insulin release if at the same time the initiator site is whether the spectrum of secretory responses observed activated; then, via co-operative interaction between in vitro is applicable to the in vivo situation. We have the units, a faster rate of insulin release occurs than in tested the effects of intravenous injection of initiators the absence of potentiator. Whether the initiator site and potentiators of insulin release and of mannohep- responds directly to glucose (and other initiators) or to tulose on plasma insulin concentrations in overnight metabolites thereof formed within the B-cell has not - fasted conscious rats. 280 S.J.H. Ashcroft and J.R. Crossley: Sugar Effects on Insulin Release in vivo

Material and Methods thiolate. Rat insulin standard (Lot 170) was a gift from Dr. A.J..Moody of the Novo Research Institute, Co- Sugars penhagen, Denmark. nsI-insulin (50 ~tCi/~tg)was pur- chased from the Radiochemical Centre, Amersham. All test sugars (D-glucose, L-glucose, N.acetyl-D- Guinea pig antibovine insulin serum was prepared in glucosamine, D-fructose, D-galactose, D-mannohep- this laboratory. Insulin-free rat plasma was added to tulose, D-glucosamine HCI) were of the purest grade each tube not containing a test plasma sample. The commercially available. Glucosamine HCI was con- incubation volume was 0.2 ml; usually 20 ~tl of plasma verted to free glucosamine by shaking with triethyl- was assayed in triplicate. "Free" and antibody-bound amine/ethanol. Since the D-glyceraldehyde (Sigma, hormone were separated by addition of albumin-coat- G-4876) contained 30% of an unidentified impurity ed charcoal (1 ml 0.6% w/v Norit GSX charcoal in DL-glyceraldehyde (British Drug Houses) was used diluent containing 0.4%, w/v bovine albumin). The for most experiments. Similar results were obtained tubes were centrifuged, the supernatant removed by with both sugars. Solutions for injection (1 ml) were suction and discarded, and the 125I associated with the prepared in sterile saline immediately prior to use. charcoal pellet measured using a solid well-type gamma radiation counter. Insulin concentrations were Animals evaluated using a computer programme based on the function described by T~ljedal and Wold [11]. Male albino Wistar rats (230-280 g) were used throughout. Rats were allowed free access to food and Assay of Plasma Sugars water prior to surgery. Cannulae (2FG, Portex, Kent, England) were inserted into the left femoral vein and Plasma glucose concentrations were measured by artery under Nembutal anaesthesia, secured by surgi- the glucose oxidase method [12]. Plasma N-acetylglu- cal thread, and kept patent with saline containing 160 cosamine concentrations were measured by two me- U/ml heparin [8]. After recovery the animals were thods. One of these utilised N-acetylglucosamine ki- kept in restraining cages overnight, with food withheld nase prepared from rat liver by the method of Saeki et but free access to water. al. [13]. 5~tl plasma wasadded to 1.5 ml 150 mlTRA Test substances were injected into the femoral vein; pH 7.5 containing 10 mM MgCI2, 6 mM ATP, 5 mM nine arterial blood samples (0.35 ml) were collected at phosphoenolpyruvate, 0.1 mM NADH, 1 U/ml pyru- the times stated in the figures and tables and immedia- vate kinase, 1 U/ml lactate dehydrogenase and a suit- tely chilled in ice. Theplasma separated by centrifuga- able volume of N-acetylglucosamine kinase. The oxi- tion was assayed immediately or stored frozen. In one dation of NADH was measured by adsorption at 340 series of experiments, the femoral vein cannula was nm. Glucose was not a substrate for N-acetyl-glucos- connected to a peristaltic pump (Pharmacia). N-ace- amine kinase. No N-acetylglucosamine could be de- tylglucosamine (1 • 10 -3 moles) was injected tected in normal plasma samples. A chemical assay of through a sideport, and then infused for 60 min at a N-acetylglucosamine [14] gave similar results. rate of 2.4 • 10 -5 moles (40 V1) per minute. A few animals which had been tested with glucose Calculation of Results or saline alone, were used for a second experiment, two hours after completion of the first. No significant In each experiment the plasma insulin and glucose difference in plasma glucose or insulin concentration concentrations at time zero for each rat were sub- (expressed relative to that at time zero for each exper- stracted from the values at the other time points. The iment) was found when consecutive identical time mean changes in glucose (AG) and insulin (AI) courses were performed with glucose, indicating that (_+ S. E. M.) for each test substance are plotted against the secretory responses were not affected by the re- time. duction in blood volume. Results Assay of Plasma Insulin The mean arterial plasma glucose and insulin con- Plasma insulin concentrations were measured by centrations at time zero in these experiments were radioimmunoassay essentially as described by Albano 5.3 + 0.1 mM and 19 + 1.4 ~tU/ml respectively et al. [9]. The assay buffer reagent diluent was (mean + S.E.M.; n = 72). In control experiments in 0.05 M sodium phosphate pH 7.4 containing 0.1% which rats received injections of saline or L-glucose w/v bovine albumin (Frn V) and 6 • 10-3M mer- (1.4 • 10- 3moles) the changes in plasma insulin and S. J. H. Ashcroft and J.R. Crossley: Sugar Effects on Insulin Release in vivo 281

Table 1. Changes in plasma insulin (AI) and glucose (A G) concentrations in response to intravenous injection atzero minutes of D-galactose, D-fructose or D-glucosamine. Results are given as mean + S.E.M. for the number of experiments stated

Monosaccharide injected (1.4 X 10 -3 moles)

Time D-Galactose D-Fructose D-Glucosamine (rains) AG(mM) AI(~tU/ml) ' AG(mM) AI(~tU/ml) AG(mM) AI(~tW/ml)

n=4 n=4 n=5 --10 0.31 + 0.22 --4.4 + 2.0 --0.56 + 0.44 --5.0 + 6.0 --0.17 + 0.22 --3.0 + 1.5 0 0 0 0 0 0 0 1 0.53 + 0.06 b 21 + 3.5b --0.28 + 0.17 239 + 38 b --0.39 + 0.28 0 + 3.0 2.5 0.71 _+ 0.29 11 + 4.0 --0.22 + 0.17 136 + 5.0 c 0 + 0.37 67 ___ 16" 5 0.83 -+ 0.40 6.5 + 2.5 1.3 -I- 0.11 b 67 q- 8.5 b 0.56 _+ 0.17" 29 _+ 15 10 1.0 + 0.32 9.0 -+ 2.5 a 3.7 _+ 0.39 b 53 + 19" 1.3 + 0.39 a -3.5 + 3.0 20 1.1 _+ 0.16 b 18 +- 5.0~ 4.2 + 0.61 t' 48 _+ 13" 2.4 _+ 0.33 b -2.0 + 3.0 40 1.3 + 0.14 b 7.0 + 7.5 2.6 +_ 0.56" 37 _+ 17 5.2 -+ 0.56 t' 0 + 3.5 60 2.2 _+ 0.29 b 8.5 + 5.5 2.1 + 0.89 a 22 _+ 20 6.9 -+ 0.83 c 6.0 _+ 4.0

a p< 0.05;bp < 0.01;Cp < 0.001

Table 2. Changes in insulin (AI) and glucose (AG) concentrations in response to intravenous injection at zero minutes of D- or DL glyceraldehyde alone or with D-mannoheptulose. Results are given as mean +_ S. E.M. for the number of experiments stated

Substance injected

Time D-glyceraldehyde DL-glyceraldehyde DL-glyceraldehyde DL-glyceraldehyde (mins) (1.4 X 10-3moles) (5 X 10-4moles) (5 X 10-4moles) (1.4 X 10--3moles) plus D-mannoheptulose (1.5 X 10-3moles) AG(mM) AI(~tU/ml) AG(mM) AI(~tU/ml) AG (mS) AI(~tU/ml) AG(mM) aI(~tU/ml)

n=3 n=3 n=3 n=5

--10 --0.29 --+ 0.02 -10 + 0.5 0.06 + 0.61 0.5 _+ 2.5 0.50 _+ 0.67 -4.5 _+ 3.0 2.9 .4- 0.11 b -14.0 + 3.0 0 0 0 0 0 0 0 0 0 1 0.07 "4- 0.14 180 _+ 35 a 0.06 _+ 0.17 175 _+ 15 b 0.06 _+ 0.39 189 + 14 b 0.06 + 0.06 67 + 23 a 2 0.44 + 0.21 114 + 12.5" 0.11 + 0.06 60 _+ 4.5 u 0 + 0.39 151 _+ 21" 0.44 + 0.06 9.5 _.+ 2.5" 5 0.09 + 0.15 6.5 + 7.5 0.17 --+ 0.17 --4.0 + 4.0 1.2 _+ 0.33 73 + 13" 0.94 ----_0.17 b -5.0 + 1.5" 10 0.09 --+ 0.21 --14 -+ 6.0 0;56_+0.17 --12 + 6.0 3.3 +0.50" 24 + 7.0 2.4 --0.33 b -11 + 3.5" 20 0.46 + 0.07" --11 +_ 6.5 1.3 +0.44 0.5+ 7.0 4.3 --+0.72" 4.0+ 8.5 3.8 +0.39 c -33.5 + 3.0 b 40 0.43 + 0.21 --10 + 5.5 1.1 +0.50 --2.0+ 2.0 4.9 +0.89 a 23 + 8.5 5.7 +0.33 c - 15 -I- 2.58 60 0.83 --+ 0.37 --6.0 + 5.0 0.94 + 0.61 --2.5+ 4.0 5.3 _+ 0.72" 33 _+ 14 6.5 --+ 0.33 c -10 + 4

"p< 0.05; b P< 0.01; Cp < 0.001

glucose concentration were not statistically significant the exception of the highest dose of glucose, plasma (with the exception of a small increase in plasma insu- insulin concentrations were restored to basal by 60 lin at time 60 min after L-glucose). mins. The effects of injection of glucose (7 • 10- 5 _ 1.4 The effects of fructose and galactose are given in • 10- 3moles) are shown in Fig. 1. Peak insulin con- Table 1. Fructose (1.4 • 10-3moles) administration centrations were observed 1 min after injection, and produced a rapid rise in plasma insulin and also hyper- there was a graded response to increasing glucose glycemia. However the peak rise in insulin occurred dose. before any change in plasma glucose i.e. within the The effect of glucose (5 • 10-4moles) was totally first 2.5 min after injection. Galactose (1.4 • 10 -3 abolished by simultaneous administration of manno- moles) administration, however had only a very small heptulose (1.5 • 10-3moles) and plasma glucose effect on plasma insulin concentration; moreover the concentrations remained elevated for 60 min. With possibility could not be excluded that the observed ~tUsgld u! oi~ugqa moqk[~ uo!laogu! ou!mgsoanlg :~m~g I mtu ~ ~s:qt oql XOAO pa:unooo uo!l~:~luaouoo u!Insu! A gms~ld jo UO.IleAOIO po~IJ~IA[ "I ~ m UOA~ OSIe k~ o:m (soIotur _ OI • #'I) ou!tuesoonI~ jo slooljo otl,L "el~p A osoql *q popDoad s! oS~alOa utlnsu ! no osologlgg jo x k~ I+1+ I+ I+ I+ I+ I+ I+ g- loot~o laoa!p g .m] O0uop.IAO OU oOUOH "u!m I om!l lg osoonlg gUls~Id u! uO!I~AOIO (~;0"0 > d) lu~o!p.ug!s q~ mq Wins aql ol alqglnq.u11~ S~A~ u!insu ! u! o~tmqo tl t~

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Alnsulin A N-Acetylglucosa rhine acetyl-D-glucosamine is given in Table 3. N-acetyl- (t~U/rnl) (mM) 200 r -26 glucosamine was a potent insulin releasing agent; this effect was not mediated by conversion to glucose since plasma glucose concentrations were consistently lowered following N-acetyl-glucosamine. There was a graded secretory response to injection of N-acetylglucosamine(1.7 • 10 -4- 1.4 X 10 - 3moles) and the response was totally abolished by mannoheptulose. 10Q 13 Plasma N-acetylglucosamine concentrations following N-acetylglucosamine injection are also given in Table 3; plasma N-acetylglucosamine had returned to basal (i. e. undetectable) 60 min after injection. The effect on insulin release and blood glucose of main- taining a raised plasma N-acetylglucosamine concen- AOlucose 0 .0 (rnM) tration by infusion is shown in Fig. 2. Insulin concentration, after an immediate peak, remained elevated until the end of the infusion of N-acetylglucosamine and a marked lowering of blood glucose concentration was observed.

N-AcGluc in, fusion J, i (" ...... ~ ...... ",...... i I --3 -10 O 20 Z,0 60 120 Discussion Time (rains.) Fig. 2. Changes in plasma insulin (Fll), glucose (I) and N-acetyl- Previous studies have established the specificity of glucosamine (&) concentrations after injection at time zero of insulin secretory responses to sugars in vitro [1, 2]. 1 X 10 -3 moles N-acetylglucosamine followed by infusion of The present results provide strong evidence that a N-acetylglucosamine (2.4 X 10 -5 moles/min from time zero to similar pattern of responses may exist in the more 60 rain. The data are mean values of 3 experiments complex in vivo situation. Thus plasma insulin con- glucose concentrations. Subsequently hyperglycemia centrations were rapidly increased by D-glucose, (but occurred without change in plasma insulin concentra- not by L-glucose), by glucosamine and by glyceralde- tion. With this dose, the response was consistently hyde, previously shown to be initiators of insulin re- delayed; no increase occurred in plasma insulin until lease in vitro [1, 2, 3]. Mannoheptulose abolished the 2.5 min after injection. With a lower dose of glucos- effect of glucose on insulin release but not that of amine, peak plasma insulin concentration was glyceraldehyde, as in vitro [2, 3]. A dual action of achieved at 1 min after injection. (results not shown). glucosamine on insulin release has been described in Table 2 shows that 5 x 10-4moles D-glyceralde- vitro i. e. glucosamine itself stimulated insulin release, hyde elicited a rapid and marked rise in plasma insulin albeit weakly, but inhibited glucose - stimulated in- without significant change in plasma glucose coiacen- sulin release [2]. The kinetics of these effects have not tration. Plasma insulin concentration returned to ba- been reported. The present study shows that, at a sal 5 min after D-glyceraldehyde injection. DL-glyce- physiological glucose concentration, glucosamine ra- raldehyde was also insulinogenic: the response to pidly increased insulin release; subsequently however, 5 x 10-4moles DL-glyceraldehyde was similar to despite hyperglycemia, insulin levels remained at a that with D-glyceraldehyde. A larger dose of DL-gly- basal level, suggesting that the inhibitory effect of ceraldehyde (1.4 x 10-3moles) was hyperglycemic glucosamine predominates after the first few minutes. but the peak insulin concentration occurred before Fructose and N-acetylglucosamine potentiated in- any change in plasma glucose concentration. The insu- sulin release in vitroin the presence of sub-stimulatory lin secretion induced by 5 X 10-4moles DL-glyce- glucose concentrations [1, 2, 4], and both sugars are raldehyde was not abolished by the simultaneous ad- shown here to produce marked and rapid increases in ministration of 1.5 x 10-3moles mannoheptulose plasma insulin concentrations in vivo. The initial se- although the peak insulin concentration achieved was cretory response to fructose injection may be a direct lowered (one animal which failed to respond to glyce- effect of fructose on the B-cell: from 5 min after raldehyde and mannoheptulose has been excluded fructose injection pronounced hyperglycemia occurs from these data). which itself will influence insulin release. The strong The insulin secretory response to injection of N- effect of N-acetylglucosamine cannot be attributed to 284 S.J.H. Ashcroft and J.R. Crossley: Sugar Effects on Insulin Release in vivo its conversion to glucose; plasma glucose levels show Dr. Crossley is the recipient of a New Zealand University Grants indeed a tendency to decrease slightly after N-acetyl- Committee Post-doctoral Fellowship, and a New Zealand Medical glucosamine injection, presumably in response to the Research Council Travel Grant. hyperinsulinemia. That hypoglycemia is not more marked despite the hyperinsulinaemia may perhaps References indicate some interference by N-acetylglucosamine 1. Ashcroft, S.J.H., Bassett, J.M., Randle, P. J.: Insulin secretion with peripheral or hepatic glucose metabolism. How- mechanisms and glucose metabolism in isolated islets. Diabetes 21, Suppl. 2, 538-545 (1972) ever when N-acetylglucosamine was infused to main- 2. Ashcroft, S.J.H., Weerasinghe, L. C. C., Randle, P.J.:Interre- tain a constant blood concentration of about 20 mM, lationship of islet metabolism, adenosine triphosphate content marked hypoglycemia was observed. It is also of inter- and insulin release. Biochem. J. 132, 223-231 (1973) est that the N-acetylglucosamine tolerance curves are 3. Hellman, B., Idahl, L.-A., Sehlin, J., T~iljedal, I.-B.: The pan- similar to equimolar glucose tolerance curves; wheth- creatic ~cell recognition of insulin secretagogues. Comparisons of glucose with glyceraldehyde isomers and dihydroxyacetone. er insulin increases N-acetylglucosamine utilization Arch. Biochem. 162, 448-457 (1974) is not known. Mannoheptulose abolished the effect of 4. Curry, D.L.: Fructose potentiation of mannose-inducedinsulin N-acetylglucosamine in vivo as it has been shown to secretion. Amer. J. Physiol. 226, 1073 - 1076 (1974) do in vitro [2]. 5. Pfeiffer, E.F., Ziegler, R.: In: Handbuch des Diabetes Mellitus. (ed. E. F. Pfeiffer), Vol. 1, p. 221. Lehmanns Verlag, M/inchen, It was not possible with the present technique to 1969 demonstrate a direct effect of galactose on insulin 6. Pozza, G., Galansino, G., Hoffeld, H., Foa, P.P.: Stimulation of release. Although injection of 1.4 x 10-3moles ga- insulin output by monosaccharides and monosaccharide deriva- lactose elicited a small but significant increase in tives. Amer. J. Physiol. 192, 497-500 (1958) plasma insulin this might have been caused by the 7. Karam, J.H., Grasso, S.G., Wegienka, L.C., Grodsky, G.M., Forsham, P.H.: Effect of selected hexoses, of epinephrine and small but significant rise in plasma glucose. Morever of glucagon on insulin secretion in man. Diabetes 15, 571-78 the magnitude of the observed insulin response was (1966) close to the limits of the method, as assessed in control 8. Aynsley-Green, A., Biebuyck, J.F., Alberti, K. G. M. M.: An- animals. aesthesia and Insulin Secretion: the effects of diethyl ether, Although clearly with an in vivo study the presence halothane, pentobarbitone sodium and ketamine hydrochloride on intravenous glucose tolerance and insulin secretion in the of plasma glucose precludes differentiation between rat. Diabetologia 9, 274- 281 (1973) initiators and potentiators of insulin release, the pre- 9. Albano, J.D.M., Ekins, R.P., Maritz, G., Turner, R.C.: A sent results are in good overall agreement with in vitro sensitive precise radioimmunoassay of serum insulin relying on observations. That the highly specific insulin secretory charcoal separation of bound and free hormone moieties. Acta. endocr. 70, 487-509 (1972) response should extend to the naturally occurring glu- 10. Hales, C. N., Randle, P. J.: Immunoassay of insulinwith insulin- cosamine and N-acetylglucosamine (indeed the latter antibody precipitate. B iochem. J. 88, 137-137 (1963) appears at least as potent as glucose, in vivo) could 11. T~iljedal, I.-B., Wold, S.: Fit of some analytic functions to indicate a role for these sugars in the regulation of insulin radioimmunoassay standard curves. Biochem. J. 119, 139-143 (1970) insulin release. Isolated islets have been shown to be 12. Huggett, A.St.G., Nixon, D.A.: Enzymic determination of capable of metabolising glucosamine and N-acetylglu- blood glucose. Biochem. J. 66, 12P (1957) cosamine [2]; extracts of rat islets contain N-acetyl- 13. Saeki, H., Suzuki, R., Tsuiki, S.: Purification and properties of glucosamine kinase (unpublishedobservations). How- N-acetylglucosamine kinase from rat liver. Tohoku J. exp. Med. ever under what circumstances, if any, these sugars 105, 87-88 (1971) 14. Reissig, J. L., Strominger, J.L., Leloir, L.F.: A modified colori- are available to the B-cell is not known. metric method for the estimation of N-acetylamino sugars. J. biol. Chem. 217, 959-966 (1955) Acknowledgements. The authors gratefully thank Prof. Dr. S. J. H. Ashcroft K. G. M. M. Alberti and Dr. Paul Holloway, Southampton General Dept. of Biochemistry Hospital for instruction in the surgical techniques, Dr. Paul England Medical School for writing the computer programme for insulin immunoassay, and Univ. Walk Mrs. R. Rawson for skilled technical assistance. These studies were Univ. of Bristol supported by grants from the British Diabetic Association, the Bristol. BS8 1 TD British Insulin Manufacturers and the Medical Research Council. England