PROGRESS IN ENDOCRINOLOGY

AND METABOLISM

Insulin Release:

The Fuel Hypothesis

W. J. Malaisse, A. Sener, A. Herchuelz, and J. C. Hutton The

immediate

release

and

direct

by circulating

is thought

to be mediated

a sequence

regulation

nutrients,

of

especially

in the pancreatic

of metabolic,

insulin glucose,

B-cell by

ionic, and motile

events.

On the basis of previous work, it is assumed that the process by which glucose is recognized otropic

agent

changes

evoked

Several dates

entirely

factors for

the

depends

by the

coupling

handling. nucleotides

(adenosine-

monophosphate),

or

nucleotides.

account

for

Ca’+-Ca*+

of a native

dose-response

curves

depicts

the

of

glucose

concentrations.

rate

between

operative

in

in H+

glucose-induced outflow

rate, all

hyperbolic-like

exchange

in affinity

nutrients

the

dose-

values at noninThe changes

in

may account for a glucose-

pattern

process

insulin

for

these

release

a

parameters

at

increasing that such a

and cationic

to

to

system.

to that which

It is proposed

metabolic

due

ionophoretic

analogous

response

other

events

is

insulinotropic

and that its time course may be relevant to

phasic

aspect

nutrient-induced other

the

changes

glucose concentrations.

yield a sigmoidal

the

The

displaying

concentration

coupling

However,

H+ and

sulinotropic induced

5’-cyclic

could play a modulatory

insulin release.

curves with half-maximal

NAD(P)H

in the

and cyclic

guanosine-3’.

in K+ and Ca2+ fractional

response

The

changes

of two essential coupling factors:

parameters

change

that

and calcium

on the

may

decrease

metabolic

intermediates

or both,

pyridine

fluxes

these

cells. candi-

of insulin release seems to depend

generation reduced

islet

as possible

between

of glycolytic

role upon stimulated

metabolic

in the

ionic events such as altered

It is acknowledged

initiation

as an insulinthe

chloride, sodium, potassium,

concentration

three

sugar

are considered

changes and subsequent phosphate,

on

pancreatic

the regulation

of

insulin

release

of

hormones).

release. insulin

which

of fuel homeostasis,

the capacity of circulating

nutrients

Thus,

(and

the

possibly

is essential

would depend

for on

to act as a fuel

in the islet cells. This concept raises’s question as to the existence regulating function

and nature

of feedback

mechanisms

the metabolic fluxes in the islet cells as a

of their energy expenditure.

MAMMALIAN SPECIES, INproducing cells are grouped

the insulintogether with other endocrine cells (e.g., glucagon-, somatostatin- and pancreatic polypeptide-producing cells) in small islets, themselves scattered in the exocrine pancreas. This anatomic organization represents a major obstacle in obtaining both an abundant and a pure B-cell preparation. It may be wise, therefore, to introduce any review on B-cell physiology by reminding oneself that, Metabolism,

Vol. 28, No. 4 (April), 1979

when lo&200 individual islets are collected from a single pancreas, this represents less than 1.0 wet weight tissue, or approximately 0.1 mg of protein. The present report deals with selected biophysical and biochemical features of B-cell function, which are considered within the framework of a hypothetical model for the nutritional regulation of insulin release. THE PHYSIOLOGIC REGULATION OF INSULIN RELEASE

In vivo, the release of insulin is regulated by several factors.“2 Some of them exert a direct and immediate effect upon the pancreatic B-cell: such is the case for certain nutritional (e.g., glucose), hormonal (e.g., catecholamines and gastrointestinal hormones), and neurohumoral transmitters) factors. Other (e.g., cholinergic regulatory factors exert a delayed and either direct or indirect control upon the release of insulin:3 such is the case in certain ontogenic (e.g., perinatal period), endocrine (e.g., pregnancy), and alimentary (e.g., fasting) situations. The present review is restricted to the immediate regulation of insulin release by nutritional factors, with emphasis on the process of glucose-induced insulin release. It should be realized, however, that glucose is only one among several nutrients that may modulate the secretory activity of the pancreatic B-cell. The influence of other nutrients upon insulin release was recently reviewed.4 THE PROCESS OF GLUCOSE-INDUCED INSULIN

RELEASE

From the cytophysiologic standpoint, the process of glucose-induced insulin release can be viewed as a sequence of metabolic, cationic, and From the Laboratory of Experimental Medicine, Brussels Universiiy School of Medicine, Brussels, Belgium. Received for publication September I I, 1978. Supported in part by grants from the Fends de la Recherche Scientifique Mt?dicale, Brussels, Belgium. Address reprint requests to W. J. Malaisse. M.D., Laboratory of Experimental Medicine, I I5 Boulevard de Waterloo, B-1000. Brussels, Belgium. 01979 by Grune & Stratton, Inc. 0026~495/79/2804~012$02.00/0 373

374

MALAISSE

mechanical events.’ In a simplified manner, the metabolic events may be relevant to the process of glucose identification by the B-cell as a stimulus for insulin release?’ the cationic events, and especially the accumulation of Ca*+ in a critical site of the B-cell, can be considered as providing the trigger for insulin release:8,9 the motile events, in which a microtubular-microfilamentous system is thought to be intimately involved, are responsible for the translocation and exocytosis of secretory granules.‘&” Since each of these three major steps in the secretory sequence was recently reviewed, emphasis will be given here to more recent observations, which deal mainly with the coupling between metabolic and cationic events. THE

GLUCOSENSOR PANCREATIC

DEVICE

ET AL.

absence of extracellular glucose by provoking glycogenolysis and glycolysis from endogenous stores of glycogen.29.30 In the following discussion, it will be assumed that the process of glucose-induced insulin release is indeed entirely dependent on metabolic events evoked by the sugar in the pancreatic B-cell. The main theme of the discussion will be to establish a link between these metabolic events and subsequent events in the secretory sequence. GLUCOSE-INDUCED

IONIC

EVENTS

Before considering the several candidates for the role of “messenger” between metabolic and ionic events, it may be helpful to briefly consider which are the major ionic changes encountered in the B-cell exposed to glucose.

OF THE

B-CELL

For several years, two contrasting theories were advocated concerning the organization of the B-cell glucosensor device. According to the first theory, the molecule of glucose itself serves as a signal for insulin release by interacting with a stereospecific glucoreceptor, presumably located at the B-ceil membrane.14 According to the second theory, the signal for insulin release is generated by the metabolism of glucose in the pancreatic B-cell in the form of a metabolite or cofactor. Subtle models were even offered to reconcile both theories.“-” In our opinion, there is no evidence and no need to postulate the existence of a glucoreceptor. Nevertheless, it should be realized that a component of the B-cell boundary,‘* which is stereospecific for Dglucose,‘9.20 exists in the form of the carrier responsible for glucose transport across the plasma membrane.2’ As reviewed in greater detail elsewhere,6 the major arguments in support of the metabolic or substrate-site hypothesis** are as follows: (1) the relative insulinotropic capacity of different sugars (glucose, mannose, fructose, galactose, sorbitol, erythrose, and glyceraldehyde) correlates with their aptitude to be metabolized in the islet cells;23-26 (2) the more marked insulinotropic capacity of (YD-glucose, as distinct from &D-glucose, coincides with a higher glycolytic rate due to the stereospecificity of the enzyme phosphoglucose isomerase for a-D-glucose-6-phosphate;“.** (3) it is possible to stimulate insulin release in the

The Phosphate Flush A change in glucose concentration provokes an almost immediate and transient efflux of orthophosphate from perifused or incubated islets. This “phosphate flush” displays a graded response to increasing glucose concentrations, with a threshold at glucose 2.8 mM or more, a half-maximal response at glucose 6.7 mM, and a maximal response at glucose 1 1.1 mM.3’m’3 Cl- Handling Preliminary data suggest that glucose decreases the intracellular concentration of Cl- by increasing its fractional outflow rate.34 Kt Handling Glucose provokes a rapid, sustained, and reversible decrease in K+ efflux in the islet celis.35~38This results, under steady state conditions, in an increased concentration of 39K+, or enhanced net uptake of 42K+ (or 86Rb+) by the islets (Figs. 2B and 2F). The dose-response curve for the effect of glucose upon “Rb+ fractional outflow rate displays a half-maximal value at a glucose concentration of 2-4 mM, which is far below the threshold concentration for stimulation of insulin release (Fig. 2L). Nat Handling Glucose exerts a dual effect upon Nat handling. It causes a marked, rapid, and sustained increase in 22Naf fractional outflow rate by a mechanism which is apparently resis-

INSULIN RELEASE: THE FUEL HYPOTHESIS

tam to ouabain. It also apparently increases the Naf inflow rate, the net result of these two elTects being a modest decrease in the steady state concentration of ‘2Na’ in the islets.39 Incidentally, the cytosolic concentration of Na+ is apparently much higher in the islet than in most other cell types, amounting to 50-75 mM.39,40 Figure 1 depicts, in a simplified and speculative manner, the major effects of glucose upon the movements of monovalent cations in islet cells. Co’+ Handling It is now firmly established that glucose causes Ca*+ accumulation in the islet cells, as recently confirmed by the measurement of 40Ca’+ in the islets by atomic absorption.’ Radioisotopic4’d3 and ultrastructura144m46 studies have also documented this effect of glucose. However, it is still unclear to what extent an increase in Ca” influx, a decrease of Cali efflux, and an intracellular redistribution of Ca2+ contribute to the changes in Ca*+ handling occurring in the B-cell exposed to glucose. This and related problems are considered in greater detail elsewhere.8T9 One of the most sensitive methods to charac-

375

terize the effect of glucose upon Ca2+ handling by the islets consists in monitoring 45Ca efflux from prelabeled and perifused islets, since the effluent radioactivity originates from intracellular sites.47 The changes in 45Ca efflux seen in response to glucose do not correspond to a reuptake process, as had been postulated by Hellman.48 At high-glucose concentration, they consist of an initial decrease, and later an increase in 4’Ca efflux. These two phenomena correspond to two distinct Ca*+ movements, which are characterized by vastly different dose-action relationships (Figs. 2C and 2L) and can be dissociated from one another. They both are dependent on the integrity of glucose metabolism and represent sustained and rapidly reversible processes, the coexistence of which may cause them to be masked, in part at least. one by another.49 The glucose-induced fall in 45Ca efflux, which is selectively abolished by valinomycin (1 .O nM or more), displays a hyperbolic dose-response curve, with a half-maximal response being reached at glucose 3-4 mM. It is unaffected by inhibitors of Ca*+ influx (e.g., EGTA, Mg’+, It may represent a organic Ca’+ antagonists). primary or secondary reduction in the outwards

NO GLUCOSE

Fig. 1. Schematic view for the handling of monovalent cations by pancreatic islets incubated in the absence or presence of glucose (16.7 nM. Steady state values for pool sizes are expressed as pmole/islet (intracellular water content: 2-3 nl/isletl. Fluxes are given in the rectangles as pmole/islet/min. The opening of the gates reflect changes in permeability. as judged from either the inflow rate or fractional outflow rate of each cation. Also depicted is the existence of a small and possibly organelle-bound pool of Na+. characterized by a low turnover rate. A 312 stochiometry is postulated for Na+/K+ transport by the membrane-associated ATPare. and all influent K+ is assumed to be transported by such an ATPase.37.3e

GLUCOSE lb 7 mM

MALAISSE ET AL

376

ADENYLATE CHARGE

SYNTHESIS

loo-

NET

:

UPTAKE

45Ca EFFLUX

I

‘INSULIN RELEASE /

0

5.6

11.l

16.7

0

5.6

GLUCOSE

11.1

16.7

0

5.6

11.1

16.7

(rnM)

Dose-response curves for the effects of glucose upon metabolic, cationic, and functional parameters in isolated Fig. 2. pancreatic rat islets. The results (mean f SEMI refer to the glucose-induced increments above basal value (no glucose), and are expressed in percent of the mean increment found within the same experiment(s) at glucose 16.7 mM. Also shown is the glucose concentration corresponding to a 50 % increment relative to the reference value found at glucose 16.7 mM. (A) The concentration of ATP. ADP, and AMP was measured in isolated islets after 30 min of incubation at different glucose concentrationsM During this period, the ATP concentration lowers in islets incubated in the absence of glucose.79.‘07 As little as 2.8 mM glucose (but apparently not 1.7 mMl is sufftcient to prevent the decrease in adenylate charge (i.e., (ATP + 0.5 ADP]I[ATP + ADP + AMP]), which averaged 0.889 f 0.007 In = 148). 0.783 ? 0.012 (n = 65). 0.773 ? 0.011 (n = 23) and 0.775 rt 0.008 In = 89) in the absence of glucose, and at glucose, 2.8-5.6 mM, 8.3 mM. and 16.7 mM, respectively.” Of all the curves depicted in this figure, that characterizing the changes in adenylate charge yields the lowest apparent “Km.” Thus, the glucose-induced increment in adenylate charge reaches one half of its maximal value at a glucose concentration of approximately 2.0 mM. The panels A-M are arranged in an order that corresponds roughly to the increasing values for such an apparent “Km.” (6) Glucose provokes a rapid, sustained, and rapidly reversible decrease in ‘*K+ or “Rb’ fractional outflow rate, a decrease that may contribute to B-cell depolarization. Under steady state conditions, the fractional outflow rate of “Rb+ decreases from 5.86 -t 0.14 (n = 40) in the absence of glucose to 2.88 f 0.09 (n = 9) in the presence of glucose, 16.7 mM. The results obtained by either Henquin (open circles’s) or Malaisse et al. (closed circles57) demonstrate that the effect of glucose upon K+ conductance cannot be considered as a none-or-all mechanism. Half-maximal changes are seen at glucose 2.2-3.5 mM. (Cl When islets are prelabeled with 4sCa2+ and first perifused in the absence of glucose, the administration of glucose provokes a rapid, sustained, and rapidly reversible decrease in “Ca’+ fractional outflow rate.“’ At high-glucose concentrations. this effect may be masked by a secondary increase in 46CaZ+ efflux.*’ The closed circles refer to (Caption continued on next page)

NSULIN

RELEASE: THE FUEL HYPOTHESIS

377

the difference in -Ca effhrx between the level found 3 min after introduction of glucose at the stated concentration and the control level found et the same time in experiments carried out in the absence of glucose throughout the perifusion period. The open circles refer to a complementary phenomenon, in that the differences in l‘Ca efflux between control and experimental values refer to those seen 3 min after increasing the glucose concentration from the stated velue up to 16.7 mM, and ere plotted with the xero point at the top and the 100 % -point et the bottom of the ordinates.” Note that e glucose concentration of approximately 3.7 mM is sufficient to cause a decrease in 4sCa2+efflux, representing 60 % of that seen at glucose 16.7 m&f. (D) The output of lactic acid (pmole/90 minlislet) from the islets increases from a basal value of 23 f 1 (n = 111 to 181 + 6 (n = 102) in the presence of glucose 16.7 m&f.- The dose-response curve is not clearly sigmoidal. A striking feature is that a dramatic and dose-related increase in lactate output is observed in the 0-5.6-m&f range of glucose concentrations.” The results depicted in ID) may account, in part et least, for e glucose-induced increase in H+ output from the islets (Melaisse and Velverde, unpublished observations). This in turn may be responsible for changes in K+ and Ca*+ (phase 1) outflow. The lactate output observed at glucose 16.7 mM does not represent a maximal value, being further increased at a glucose concentration of 27.8 mM. This is also the ~88%for several other parameters. illustrated in Fig. 2; namely, for the net rate of proinsulin biosynthesis,“’ the net uptake of “Ca” and release of insulin,4’ and the utilization of [5-‘H] glucose and oxidation of [U-“C] glucose.” (El The effect of glucose upon the incorporation of ‘H-leucine in insulin-like immunoreactive peptides (‘H-65) was calculated from either the absolute values for ‘H-IRI (closed circles) or the ratio of JH-IRI/TCA-precipitable ‘H-peptides (open circles), the latter ratio being representative of the preferential effect of glucose upon the synthesis of proinsulin, as distinct from other islets’ peptides.“’ The dose-response curve for ‘H-IRI biosynthesis represents the first example of 8 truly sigmoidal curve. The concentration of glucose must reach a value between 2.5 and 3.9 mM in order to stimulate proinsulin biosynthesis. This threshold value is much lower than that required for stimulation of insulin release (i.e.. between 4.2 and 5.6 m/HI. Note that the ratio of SH-IRl/TCA-precipitable “H-paptides reaches its maximel value at glucose concentrations Lea 8.3 mM) lower than those required to cause a maximal incorporation of ‘H-leucine in ti-IRI (more than 16.7 m/H). (Fl The steady state concentration of **Rb+ expressed 8s K+ with the same specific activity as that of the incubation medium (BeRb+/5sK+) averaged 255 + 9 (n = 93) and 430 + 14 (n = 94) pmolelislet at glucose 0 and 16.7 mM, respectively. The values obtained at intermediate glucose concentrations, although derived from a smaller number of individual measurements (n = 13-14). are in fair agreement with the data shown in (8). Indeed, th8 estimated inflow-outflow rate of K+ (taken as the product of *‘Rb+ net uptake by “Rb+ fractional outflow rate) was not significantly affected by glucose, averaging 14.4 t 0.9, 11 .l i 1.2. and 12.3 + 0.8 pmole/min/islet at glucose 0.2.8. and 16.7 m&f, respectively. This suggests that the glucose-induced increment in K+ concentration in the islet cells is due mostly, if not exclusively, to the chsnge in K+ conductance.“.” (G) The data for ‘6Ca2t net uptake (pmole/90 minlislet) were obtained by either the washing technique.’ or oil sep8ration procedure.‘.” Although the net uptake of “Ca” evoked by glucose (16.7 mM), relative to b8S8f value. is somewhat higher in the washing technique (ratio of stimulated to basal uptake: 4.06 + 0.22) than the oil separation procedure (ratio of stimulated to basal uptake: 2.45 -t 0.15). the increments in *‘C8’+ net Uptake seen 8t increasing glucose concentrations. relative to the reference increment found at glucose 16.7 m&f, are comparable in both methods: they averaged 30.5 % and 26.6 % at glucose 5.0-5.6 mM and 58.5 % and 60.9 % at glucose 8.3 mAf, with the washing and oil separation techniques, respectively. When compared to the curves characterizing the effect of glucose upon “Cazf efflux (C and L), the curve for ?a’+ net uptake displays mixed properties: there is a threshold value LabOva2.8 mM) for the stimulatory action of glucose upon ‘*Ca’+ net uptake, as in the case of (l-1: but the values found 8t intermediate glucose concentrations (5.0-8.3 m/HI yield en apparent “Km” (7.2 mM) in between the values illustrated in (Cl (3.7 m/HI and (L) (10.2 m/H), respectively. (H. J) The curves relating the conversion of (5-‘HI glucose to ‘H,O (glucose utilization) and RI-‘*Cl glucose to “CO, (glucose oxidation) display an almost identical pattern.” In other words, the ratio of glucose oxidation to utilization is fairly constant.*’ Since no utilization or oxidation of exogenous glucose takes place in a glucose-free medium, the dose-response curves are characterized. in the O-4.3 mM range of glucose concentrations, by a first dose-related increase tending, with an apparent Km of 0.4 mM or less, towards a first saturation value: at 4.3 mM, the rates of glucose utilization and oxidation apparently plateau at respectively 26.9 f 3.4 % and 26.6 f 3.6 % of those seen at glucose 18.7 mM However, at higher glucose concentrations. a reescension in metabolic fluxes is noted, giving an overall sigmoidal appearance to the dose-response curves? relative to the reference value found at glucose 16.7 mM. the 50 % level ~8s reached 8t glucose 6.2-7.0 mM. At glucose 16.7 mM.the rates of glucose utilization and oxidation averaged 129 * 8 (n = 78) and 25 f 2 (n = 73) pmole/90 min/islet, respectively.‘r The latter values do not correspond to the maximal metabolic Rux. which is reached at higher glucose concentr8tions.” (K) The curv8 illustrates the glucose-induced changes in the total concentration of reduced pyridina nucleotides (NADH and NADPH) in islets exposed for 30 min to increased glucose concentrations (pooled data obtained by the enzymatic cycling and luciferase techniques?. At glucose 16.7 m/H. the concentration of NAD(P)H is approximately 1.6-1.9 times greater than the b8Sal vslue found in islets incubated in a glucose-free medium. The dose-response curve is characterized by 8 steep slope for the range of glucose concentrations above threshold (i.e.. 5.6 mMor more) and below close-to-maximal (i.e., 11 mMor less) values. with 8n apparent “Km” close to 7.4 m/H. (L) At normal extracellular Ca2+ concentration. glucose provokes 8 secondary increase in lrCa efflux.“’ The difference in “Ca efflux between the nadir value seen shortly after introduction of glucose and the later zenith value was observed in islets parifusad first in the absence of glucose and then exposed to glucose at the stated concentration. Note the similarity between the curves depicted in (K1and (l.). respectively. (64) The data for insulin release are pooled from two series of observations.“.7E The dose-response curva is typically sigmoidal. No significant effect of glucose is detected up to a 4.2 mM glucose concentration. Relative to the reference value found at glucose 16.7 m/H, s 50 % secretory response is reached st glucose 10.3 mM, which is the highest apparent “Km” illustrated in this figure. Since the maximal secretory rate is not observed 8t glucose 16.7 m&f, but at higher glucose concentrations (27.8 m/H or more), the true half-maximal response also occurs at 8 higher glucose concentration than the quoted “Km” value.“‘x The comparison of (A) to (MI clearly indicates that insulin release, which, for the animal taken as a whole, represents the sole relevant parameter of B-cell function. is Org8niZ8d so that a series of “threshold” phenomena take8 place in the regulation of metabolic parameters as a function of the extracellular glucose concentration, in the coupling between metabolic end cationic events, and in the secretory response to intracellular calcium. Such 8 functional organization is well suited to protect against inappropriate insulin release at low concentrations of circulating nutrients.

MALAISSE ET AL.

378

transport of 45Ca across the plasma membrane. The later increase in 45Ca efflux displays a sigmoidal dose-response curve, with a halfmaximal response at glucose 10 mM. Its magnitude is proportional to the rate of Ca2+ entry into the islet cells. It may represent a Ca-Ca exchange process and, as such, it could result from an increase in the ionophoretic capacity of the system mediating Ca2+ inflow (and part of Ca2+ counter-transport) in the islet cells. In conclusion, glucose provokes a dramatic and extensive remodeling of ionic fluxes in the islet cells. We are impressed that several of these changes in ionic fluxes concern movements that may not consume ATP and might be mediated by native ionophoretic systems.’ GLUCOSE

METABOLITES FOR INSULIN

AS

THE

SIGNAL

RELEASE

One implication of the substrate-site theory for insulin release could be that a metabolite of glucose acts as the signal for distal steps in the secretory sequence. For instance, the possible role of phospho-enol-pyruvate in such a perspective was recently emphasized.50.5’ However, since cY-ketoisocaproate, the metabolism of which is unrelated to that of glucose, is as potent as glucose in stimulating insulin release and, within the limits of actual knowledge, reproduces all the effects of the sugar upon cationic fluxes and bioelectrical activity,52 it is hard to believe that the specific pathways for glucose and cu-ketoisocaproate metabolism, respectively, provide a unique metabolite that would act as the key coupling factor between metabolic and cationic events. CYCLIC

AMP

AS

INSULIN

THE

SIGNAL

FOR

RELEASE

Several investigators have failed to detect any effect of glucose upon the concentration of cyclic AMP in isolated islets.s3-‘6 Other investigators have consistently observed a significant increase.574o As a rule, the latter effect is rather modest when the experiments are performed in the absence of a phosphodiesterase inhibitor. Hellman and his colleagues suggested that the effect of glucose to increase cyclic AMP concentration was the consequence of the metabolism of glucose in islet cells.57 Cerasi and Grill, who are among the most insistent defenders of the

role of cyclic AMP in the normal process of glucose-induced insulin release, do not rule out such a conclusion.6’.62 In our opinion, there is a major objection to postulate that cyclic AMP represents the key messenger in the process of glucose-induced insulin release. Thus, the third requirement defined by Sutherland and associates to establish a role for cyclic AMP as a mediator in biologic processes is not satisfied in the case of insulin release. This requirement states that the biologic response should be reproduced by (exogenous) cyclic AMP.63 In the absence of glucose or at low-glucose concentrations, the administration of exogenous cyclic AMP or an increase in the concentration of endogenous cyclic AMP fail to reproduce the effect of glucose upon islet function. For instance, when dibutyryl-cyclic AMP is used in a concentration sufficient to enhance glucose-stimulated insulin release,64 or when inhibitors of phosphodiesterase are used to increase the endogenous level of cyclic AMP up to a level greater than that normally seen at high-glucose concentrations,53354 no sustained stimulation of insulin release can be detected.29,“4 In the present state of knowledge, we would rather consider that any effect glucose may exert on the concentration of cyclic AMP in the islet cells would result in an enhancement (or potentiation) of the secretory response, which itself would be initiated by other signal(s). In other words, the glucose-induced increase in cyclic AMP concentration may contribute to the B-cell response to glucose, without being the essential factor for initiation of insulin release. Anyhow, the effects of dibutyryl-cyclic AMP or theophylline upon the handling of 45CaZ+ by the islets are vastly different from those evoked by glucose.64.h5 Hence, the latter effects can hardly be attributed solely to changes in the concentration of the cyclic nucleotides. THE

POSSIBLE

ROLE

OF PYRIDINE

NUCLEOTIDES

Almost every pathway of glucose metabolism (e.g., sorbitol pathway,66 glycolysis,25 pentose shunt,67 glycogen synthesis and breakdown with the assumption of heterogeneity in the glucose6-phosphate ~001,~~glucuronic pathway,69 Krebs cycle”) has been proposed as the key component

INSULIN RELEASE: THE FUEL HYPOTHESIS

of the B-cell glucosensor device. Likewise, each cofactor generated by the metabolism of glucose, such as ATP or NAD(P)H,7’-74 has been considered as a possible determinant of the secretory response. The idea that the availability of reduced pyridine nucleotides plays a critical role in the stimulus-secretion process has gained support from recent studies, in which agents such as menadione75~76 or NH: ” were used to decrease the concentration of NAD(P)H in the islets. For instance, in islets exposed to glucose Il. 1 mM and menadione 0.01 mM, a decrease in the concentration of reduced pyridine nucleotides was found to be associated with abnormalities of both Ca’+ handling (especially a reduction in the glucose-induced secondary rise in 45Ca2+ efflux) and subsequent insulin release.75.76 These changes occurred despite the fact that the rate of glycolysis, the output of lactate and pyruvate, the oxidation of glucose, and the concentration of adenine nucleotides were all unaffected.75.76 The existence of a causal relationship between changes in NAD(P)H concentration and Ca2+ handling is supported by the observation, under a variety of nutritional conditions, of a proportionality (Fig. 3) between the islet content in reduced pyridine nucleotides and the net uptake of 4sCa2+, which is a measurement of the exchangeable Ca2+ pools in the islets.9’78 Moreover, the dose-response curve for the glucoseinduced increase in NAD(P)H concentration is typically sigmoidal (Fig. 2K), and closely resembles that characterizing the secondary rise in 45Ca2+ efflux (Fig. 2L). We have already mentioned that the latter phenomenon could be due to a change in affinity for Ca*+ of the ionophoretic system mediating the entry of Ca*+ into the islet cells. This hypothesis is compatible with the finding that the functional capacity of certain ionophores (e.g., X537A) is indeed increased when the ionophoretic molecule is converted to its reduced derivative (Fig. 4B). A SEARCH FOR ANOTHER MESSENGER

There is an almost total dissociation between the dose-response curve characterizing the effect of glucose upon the concentration of NAD(P)H in the islets (Fig. 2K) and the Kf or CaZf (phase 1) fractional outflow rate (Figs. 2B and 2C). These cationic parameters may thus be

379

% 100

. .

50

.

/// ./: /

0

I

I

100

. /J

50

0



L

0

/

1

I

.

I

100

50 NADH

.

.’

+

%

NADPH

Correlation between the mean values for the Fig. 3. total concentration of reduced pyridine nucleotides (fmole/islet at min 30 of incubation) and the net uptake of ‘%Za (pmolelislet after SO-min incubation). Both parameters ere expressed relative to the control value found within the same experiment(s) at high-glucose concentrations (16.7 mM or 27.6 mm. The data are taken from three independent studies concerned, respectively. with the effect of menadione (upper panel’*), NHf (middle pane17’),and pyruvate (lower pan& upon islets incubated at different glucose concentrations.

regulated by other metabolic factor(s). At noninsulinotropic concentrations of glucose, i.e., the range of concentrations in which K+ and Ca*+ (phase 1) fractional outflow rates are indeed dramatically affected, several changes in metabolic parameters are observed. First, in the range of glucose concentrations between 1.7 and 4.2 mM, a significant amount of exogenous glucose is metabolized in the islet

MALAISSE ET AL.

380

B

IO-

a6-

5 io.E. 4-

t; 0” 5-

2-



4

1’

6769 PH

8

0

0.1

0.2

w/ml

lonophore-mediated translocation of Ca in an Fig. 4. artificial system.“’ The left panel illustrates the effect of pli upon the amount of Ca translocated in an organic immiscible phase containing X537A (1 .O mg/ml). Note that acidification of the aqueous phase inhibits Ca translocation in the same manner that it inhibits Ca transport across the organic phase in a Pressman cell.“’ The right panel illustrates the dose-action relationship for Ca translocation in the presence of X537A (open circles. solid line) and its dihydroderivative (closed circles, dotted line”*], which was kindly provided by Hoffmann-La Roche, Inc. (Nestley, New Jersey). Note that the ionophore in its reduced form is better abfe to translocate Ca (Couturier and Malaisse, unpublished observation). The aqueous phase always consisted of a Hepes buffer (25 mM: pH 7.0, except if otherwise stated) containing Na+. 123 mM; K+, 5 mM. Cl-. 120 mM. and a trace amount of ‘%a (approximately 4-5 pMI. The organic phase consisted of a toluene/butanol (7/3, v/v) mixture containing the ionophore at the stated concentration. Each point represents the mean of three individual measurements.

cells. The rate of utiliration (Le., the production of ‘H,O from [5-3H] glucose; Fig. 2H) and oxidation (i.e., the production of 14C0, from [U-‘“Cl glucose; Fig. 25) of exogenous glucose tends towards a first asymptotic value,” which represents about 25% of the metabolic rate found at a much higher glucose concentration (16.7 mM). In this range of concentration, glucose exerts no obvious effect upon the utilization of endogenous nutrients (as judged from the output of 14C0, from islets prelabeled with [Ur4C] palmitate). The rate of lactic acid output is also dramatically increased above its basal value in the same range of glucose concentrations (Fig. 2D). Second, the concentration of ATP, the ATP/ADP ratio, and the adenylate charge (i.e., [ATP + 0.5 ADP]/[ATP + ADP + AMP]) are much lower in the absence than in the presence of glucose. As little as 2.8 mM glucose is sufficient to prevent any decrease in ATP concentration and to maintain the adenylate charge at a level similar to that seen at higher

glucose concentrations. It may appear unlikely that the glucose-induced increase in ATP concentration or ATP/ADP ratio is solely responsible for the change in K+ and Ca2+ (phase 1) fractional outflow rate. Indeed, the latter cationic parameters are markedly affected by an increase in the glucose concentration from 2.8 to 5.6 mM; whereas the islet content of adenine nucleotides remains unchanged in this range of concentrations. Nevertheless, in view of the participation of ATP in active cationic transport processes, it would be unwise to rule out the existence of a link between the availability of high-energy phosphate intermediates and the movements of cations. Third, in the low range of glucose concentrations, there is a dramatic increase in the rate of H+ output from the islets.” The dose-response curve for H’ output is superimposable on that characterizing the change in ?a efflux (phase 1). If the extrusion of H+ generated by the metabolism of glucose occurs via a native ionophore, this could account for a concomitant reduction in Ca2+ outflow, as observed in artificial systems (Fig. 4, left panel). A WORKING

HYPOTHESIS

On the basis of the experimental data so far reviewed, the following simplified sequence of events could be proposed to account for the stimulus-secretion coupling of glucose-induced insulin release. (1) When the extracellular concentration of glucose is increased, an almost instantaneous equilibration of free glucose across the plasma membrane takes place.79.s’ The rate of glucose phosphorylation increases, thanks in part to the presence of a high-Km glucokinase.82 The rates of glycolysis, lactate output, glucose oxidation, and O2 consumption are also rapidly increased.80*83-ss (2) A first consequence of glucose metabolism is an increased generation of H+, which in turn may affect the (ionophoretic) transport of Kf and Ca’+ across membrane system(s) in the B-cell. This process could account, in part at least, for both the decrease in K+ conductance and decreased outflow of Ca2+ (Ca’+ efflux, phase I). (3) A second consequence of glucose metabolism is an increase in the concentration of reduced pyridine nucleotides. The redox state of the B-cetl may-affect the affinity for Ca2+ of the ionophoretic system, mediating the entry of

INSULIN RELEASE: THE FUEL HYPOTHESIS

Ca2+ in the islet cells (Ca*+ efflux, phase 2). (4) The remodeling of cationic fluxes eventually leads to the accumulation of Ca*+ in a critical site of the B-cell, and to the subsequent activation of the effector system controlling the translocation and extrusion of secretory granu1es.‘“.86.*7(5) The glucose-induced increase in 0, consumptions0~8s is compatible with the view that the generation of high-energy phosphate intermediates is also increased. Since the steady state concentration of adenine nucleotides is little affected in the 2.8-27.8-mA4 range of glucose concentrations, the increase in ATP generation is probably matched by an increased utilization of energy in the stimulated B-cell (Fig. 5). In this model, the effect of glucose upon the movements of cations and its capacity to serve as a fuel cannot be easily dissociated from each another, since both eventually depend on the metabolism of the sugar. Another feature of this model is that the essential coupling factors (Hf, NAD(P)H) and the major permissive factor (ATP) for insulin release are not specifically related to the metabolism of glucose. In this respect, any nutrient able, through its metabolism, to provide protons, reducing equivalents, and energy should also be able to mimic the effect of glucose upon insulin release. SIMILARITY

BETWEEN THE EFFECTS OF

GLUCOSE AND OTHER INSULINOTROPIC NUTRIENTS

The latter postulate is presently under investigation. We have recently observed that the insulinotropic actions of pyruvate” and lactateB9 are

Fig. 5. Schematic view for the coupling between metabolic and cationic wants in the process of nutriantinduced insulin release.

381

indeed compatible with the “fuel hypothesis.” The lack of parallelism between the respective oxidation rate of different nutrients (e.g., glucose, pyruvate, lactate) and their insulinotropic capacity was attributed to their unequal interference with the oxidation of endogenous nutrients. The results of an extensive study, still in progress, on the metabolism of amino and keto acids (especially leucine and a-ketoisocaproate) in pancreatic islets seem so far also compatible with the present model.s2 THE TIME COURSE FOR INSULIN RELEASE

Any model for the stimulus-secretion coupling of glucose-induced insulin release should be able to account for the dynamics of the secretory process. The release of insulin evoked by glucose and other nutrients (e.g., glyceraldehyde and a-ketoisocaproate) occurs initially as an early secretory peak, which corresponds to a long burst of spikes (approximately 1 min) in electrical records.90 Thereafter (i.e., during the second phase of insulin release), the pattern of electrical activity changes to either regular and transient depolarizations of 10-l 5 mV, reaching a plateau from which fast spikes with amplitudes of up to 25 mV start, or an almost continuous spike activity (Fig. 6). The latter pattern is only seen at very high glucose concentrations (16.6 mM or greater). The time course for insulin release, and especially its biphasic aspect, has been ascribed to a functional segregation of secretory granules within the B-ce11.9’-94Although such a functional segregation is not denied, the fact that the profile of insulin release displays specific and vastly dissimilar patterns in response to different secretagogues (e.g., glucose, sulfonylurea, and Ba *+ 9’.92,95)suggests that other factors may also participate in the precise chronological control of insulin release. Whether our model may account for all of these temporal aspects remains to be established. Nevertheless, the following considerations should be underscored. (1) In response to a square wave increase in glucose concentration, the changes in the net uptake of glucose, output of lactic acid, and concentration of reduced pyridine nucleotides occur promptly enough to account for the initial secretory response to glucose.80.84 As a matter of fact, there exists a

MALAISSE ET AL

382

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A TELEOLOGIC VIEW The “fuel hypothesis” here under consideration presents some attractive features in a teleologic perspective. Insulin represents, above all, the hormone of fuel storage.99 According to the

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chronological hierarchy among metabolic, cationic, and secretory events during the first few minutes of exposure to glucose.” (2) No definite evidence has yet been produced to indicate that early events in the secretory sequence display a biphasic pattern similar to that characterizing the bioelectrical or secretory responses. (3) Our model postulates that metabolic events regulate cationic and hence, bioelectrical and secretory events. Even in acellular systems, glycolysis and mitochondrial metabolism may display spontaneous osciliations.97’98 As far as glycolysis is concerned, the oscillatory pattern is lost at highglucose concentrations9’ It is tempting, therefore, to consider that oscillations in metabolic pathways may represent the pacemaker for oscillation in cationic and secretory events6

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present hypothesis, insulin would be released in response to an increase in the concentration of circulating nutrients by a process that would itself precisely depend on the capacity of these nutrients to act as a fuel in the pancreatic B-cell. The hypothesis could even be extrapolated to the nutritional regulation of glucagon and pancreatic polypeptide release. For instance, the release of glucagon, which shares with that of insulin a preferential sensitivity towards the Nanomer of D-glucose,‘OO.‘O’may be modulated by such nutrients as glucose and a-ketoisocaproate in a manner comparable (but in opposite direction) to that characterized in the case of insulin release (Leclercq-Meyer & Malaisse, unpublished observations). Since the different types of endocrine cells are functionally coupled within each islet,‘02~‘04It is tempting to believe that they use the same device to identify the same tropic nutrients. The action of insulin and glucagon upon their target cells plays an essential role in fuel homeostasis. Pancreatic polypeptide was recently considered as a possible satiety factor.“’

INSULIN

RELEASE: THE FUEL HYPOTHESIS

383

The islets of Langerhans could thus be viewed as a coordinated population of endocrine cells sharing both a common sensor system, to appreciate the actual availability in circulating nutrients, and a common function, to regulate in different target organs the input and uptake of such nutrients. THE

UNKNOWNS

OF INSULIN

RELEASE

Several techniques, such as the measurement of 0, uptake,85,‘06 the estimation of ATP turnover rate,‘“‘.“* and the calorimetric method”’ have indicated that glucose enhances the generation of high-energy phosphate intermediates in the islets. However, at the present time, almost nothing is known concerning the energy expenditure of the islets. For instance, the relative contribution of active cationic transport, biosynthetic processes, and motile events in the overall consumption of energy by the islet cells remain to be assessed. Only limited information is avail-

able concerning the ATPase(s) involved in these energy-requiring processes.“‘,“’ It is often considered that the energy requirement of a given cell, in a given physiologic situation, exerts a feedback control on the rate of nutrient utilization. Maybe the most intriguing aspect of B-cell physiology consists in the identification of those feedback regulatory factors that may control the utilization of glucose and other nutrients. In other words, more attention should be paid to the mechanism(s) that regulate, at a given ambient concentration of nutrient, the metabolic flux, so that the latter eventually and appropriately controls the rate of insulin release. In this and several other respects, insulin release remains an unsettled topic.15

ACKNOWLEDGMENT We thank Mrs. C. Demesmaeker secretarial help.

and S. Procureur

for

REFERENCES I. Malaisse WJ: Hormonal and environmental modification of islet activity, in Steiner DF, Feinkel N (eds): Endocrine Pancreas. Washington, The American Physiological Society. 1972, pp 237-260 2. Lambert AE: The regulation of insulin secretion. Rev Physiol Biochem Pharmacol 75:97-l 59, 1976 .3. Malaisse WJ. Malaisse-Lagae F: Influences nutritionnelles et hormonales chroniques sur la fonction insulaire, in Journees Annuelles de Diabttologie de I’Hotel-Dieu, Flammarion (Paris), 1969. pp I3 1-I 38 4. Malaisse WJ, Sener A, Hutton JC: Immediate and direct effects of nutrients upon insulin release. in Recheigl M (ed): Handbooks in Nutrition and Food. West Palm Beach. C R C Press (in press) 5. Malaisse WJ, Sener A, Herchuelz A, et al: Sequential events in the process of glucose-induced insulin release. Excerpta Med ICS 413:95-102, 1977 6. Malaisse WJ: The possible link between glycolysis and insulin release in isolated islets, in Lindenlaub E (ed): Diabetes Research Today. New York. FK Schattauer Verlag, 1977. pp I9 l-206 7. Sener A, Malaisse WJ: The metabolism of glucose in pancreatic islets. Diab Metabol (Paris) 4:127-133, 1978 8. Malaisse WJ, Herchuelz A, Levy J, et al: Insulin release and the movements of calcium in pancreatic islets, in Carafoli E, Clementi F, Drabikowski W. Margreth A (eds): Calcium transport in contraction and secretion. Amsterdam. North-Holland, 1975, pp 221-226 9. Malaisse WJ, Herchuelz A, Devis G, et al: Regulation of calcium fluxes and their regulatory roles in pancreatic islets. Ann NY Acad Sci 307:562-%X I, 1978 IO. Malaisse WJ. Malaisse-Lagae F, Van Obberghen E,

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of insulin

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A still

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MALAISSE

ET AL.

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RELEASE: THE FUEL HYPOTHESIS

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385

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Insulin release: the fuel hypothesis.

PROGRESS IN ENDOCRINOLOGY AND METABOLISM Insulin Release: The Fuel Hypothesis W. J. Malaisse, A. Sener, A. Herchuelz, and J. C. Hutton The immedi...
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