Thesis
Reference
The mechanisms of abnormal glucose uptake as studied in the heart of the insulin resistant genetically obese (fa/fa) rat
ZANINETTI, Daniel
Abstract
Le but de ce travail est l'étude de l'insulo-résistance dans le coeur perfusé du rat génétiquement obèse (fa/fa) et de son contrôle mince.
ZANINETTI, Daniel. The mechanisms of abnormal glucose uptake as studied in the heart of the insulin resistant genetically obese (fa/fa) rat. Thèse de doctorat : Univ.
Genève, 1986, no. Sc. 2232
DOI : 10.13097/archive-ouverte/unige:80317
Available at:
http://archive-ouverte.unige.ch/unige:80317
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1 / 1
Laboratoires de Recherches FACULTE DE MEDECINE
Métaboliques Professeur B. Jeanrenaud
THE MECHANISMS OF ABNORMAL GLUCOSE UPTAKE AS STUDIED IN THE HEART OF THE INSULIN RESISTANT
GENETICALLY OBESE (fa/fa) RAT
THESE
présentée ' à la Faculté des Sciences
de l'Université de Genève
pour obtenir le titre de Docteur ès sciences, mention biochimique.
par
Daniel ZANINETTI de
Genève (Suisse)
THESE No 2232 GENEVE
1986
DÉPOT DES B\BUOlHÈQUES UN\VE.HSrfA\RES
, GENÈVE) lUNIVERS11E DE
La faculté des sciences, sur le préavis .. de .. Messieur.s .. B .•.... JEANRENAU.D .•... p.rof.esseur ordinai.r.e ... e.t ... dir.ecteur. ... de. .. th.èse ... (.Lab ... r.e.che.r.ch.es .. méta.bal.ique.s ... ~ ... fac.ul.t.é de Médec.ine.)., .... J ... D.ESHU.SSES.~ ... p.r.af.esse.ur. ... o.r.dinair.e ... et. .. responsab.le .. .dev.ant ... .l.a Fa cu 1 té des ... Sc.i.ences. ... {.Dpt ... d.e ... bioctlimi.e.} ... et ... Madame ... LE ... MARCHAND.':'!BR.USIEL., ... .dac.teur
(Médecine expérimentale - Faculté de Médecine Pasteur, Nice)
autorise l'impression de la présente thèse, sans exprimer d'opinion sur les propositions qui y sont énoncées.
GEN~VE, le ... .18 .. .. décembr.e ... 19 .. 8.6 .
Le Doyen:
Jean-Pierre IMHOF Thèse
l
J
.1
·1
INTRODUCTION 10.
A. INSULIN ACTION
1. Insulin binding to its receptors 11.
2. Mediators of insulin action 12.
3. Glucose transport 15.
4. Glycolytic enzymes 26.
B. INSULIN RESISTANCE IN GENETICALLY OBESE (fa/fa) RAT
1. Syndrome of obesity 26.
2. lnsulin resistance in vivo 28.
3. Insulin resistance in muscle 29.
EXPERIMENTAL : GLUCOSE METABOLISM AND TRANSPORT IN HEART OF LEAN AND GENETICALLY OBESE (fa/fa) RATS
A. MATERIAL AND METHODS 33.
B. RESULTS
1. Glucose metabolism 43.
2. Glucose transport 51.
3. Regulation of the glucose transport system 60.
C. DISCUSSION 82.
D. CONCLUSIONS 94.
REFERENCES 96.
Le but de ce travail est l'étude de l'insulino-résistance dans le coeur perfusé du rat génétiquement obèse (fa/fa) et de son contrôle mince.
L'obesité est une pathologie progressive qui comprend deux phases principales une phase initiale sans insulino-résistance, et une phase ultérieure, où l'insulino-résistance prédomine. La phase initiale de l'obésité est la période où le dépôt de lipides dans le tissu adipeux augmente suite à des altérations du système nerveux central. Dans la phase tardive de l'obésité, le rat génétiquement obèse est caractérisé par une intolérance au glucose, une normo-glycémie et une hyperinsulinémie. Ces deux derniers points sont représentatifs de l'insulino-résistance des rats obèses car pour atteindre in vivo le même métabolisme glucidique que son témoin, le rat obèse a besoin de trois fois plus d'insuline. Pour mieux comprendre les causes de l'insulino-résistance au niveau périphérique, celle-ci a été également étudiée in vitro.
La première étape de 1' action de 1 'insuline est sa liaison avec son propre recepteur. Par conséquent une diminution de ceux-ci peut provoquer une insulino-résistance. Mais la présence de récepteurs
"d'épargne" exclut un rôle important des récepteurs à l'insuline dans l'insulino:...résistance. Le ou les défaut ( s) principal (aux) du métabolisme du glucose chez l'animal obèse se situe donc après la liaison insuline-récepteurs.
L'étude de l'insulino-résistance a été entreprise dans le coeur perfusé, pour les raison suivantes :
a) La masse musculaire est un des principaux sites de l'utilisation du glucose.
b) Les muscles squelettiques tel que le soleus utilisent 70 - 80 % de lipides pour le métabolisme énergétique, ce qui rend difficile l'observation du métabolisme du glucose.
c) Au contraire, le coeur perfusé avec 6 mM glucose et à 50 mmHg de pression utilise plus de 90 % de glucose pour le métabolisme énergétique.
d) Le coeur perfusé a un poids suffisamment grand pour rendre pratique 1 'étude des transporteurs du glucose.
Le coeur est perfusé de manière rétrograde par 1' aorte selon la méthode de Langendorff qui consiste à placer une canule en-dessus des coronaires pour irriguer tout le coeur. La pression du milieu de perfusion dans le ventricule gauche provoque les battements du coeur.
Dans un premier temps, le métabolisme total du glucose (mesuré par la différence de concentrations du glucose avant et après le coeur) a été étudié pour déterminer la présence d'une insulino-résistance. Les résultats montrent que le métabolisme du glucose est diminué dans le coeur de rat obèse, quel que soit la concentration d'insuline utilisée.
Par contre, l'action de l'insuline chez le rat obèse peut être considérée comme normale si le niveau de base du métabolisme, diminué en absence d'insuline, est pris en considération. Il n'en reste pas moins que la quantité totale de substrat que le coeur de l'animal obèse est susceptible de métaboliser par unité d'organe est bien moindre que celle métabolisée par le coeur normal.
Les intermédiaires du métabolisme du glucose, le glucose-6-phosphate, le fructose-6-phosphate, le lactate, le pyruvate et le citrate, sont élevés lors d'une utilisation accrue de lipides endogènes et exogènes ou de corps cétoniques. Toutefois, la concentration de ces métabolites du glucose ne diffère pas chez le rat obèse par rapport au rat normal (au niveau basal ou stimulé par l'insuline). Le défaut d'utilisation du glucose du rat obèse n'est ainsi pas la conséquence d'une utilisation accrue d'autres substrats (lipides), contrairement à ce que l'on observe dans certains muscles squelettiques. Ces résultats suggèrent que le défaut principal du métabolisme glucidique dans le coeur du rat obèse réside dans le transport de ce sucre à travers la membrane plasmique.
En conséquence et dans un deuxième temps, une méthode permettant d'estimer la vitesse de transport du glucose a été développée : la vitesse de transport du glucose est estimée par des mesures d'efflux de 3-0-méthylglucose marqué au carbone quatorze, un analogue du D-glucose qui est transporté, mais non métabolisé. La quantité de sucre présent dans l'espace extracellulaire est mesuré avec du L-glucose tritié, qui n'est pas transporté.
La méthode développée pour ce travail postule l'existence d'une symétrie et d'une spécificité du transport de glucose. La symétrie est confirmée par l'étude de l'influx de 3-0-méthylglucose, qui a été mesuré de la manière suivante : les ·coeurs sont perfusés en présence de 3-0-méthylglucose et de L-glucose marqués puis congelés dans de 1' azote liquide à des temps différents. Les coeurs sont digérés, et la quantité de 3-0-méthylglucose et de L-glucose est mesurée. La courbe
d'influx est identique à celle de l'efflux, cela à une concentration submaximale de 3-0-méthylglucose. La spécificité du transport pour le glucose est demontrée en mesurant une stimulation de l'efflux de 3-0-méthylglucose par le contre-transport de D-glucose, mais pas par celui du D-fructose.
Chez les rats obèses agés de 15 semaines, le transport de base est diminué de quatre fois par rapport au rat contrôle mince. L'insuline stimule le transport de 3-0-méthylglucose de manière dépendante de la concentration, mais sans jamais compenser le défaut basal. Lorsque les courbes dose-réponse de l'obèse et du normal sont superposées, il est possible d'observer une sensibilité identique à l'hormone et une diminution de la réponse maximale du coeur de l'animal obèse.
L'abaissement du transport basal de 3-0-méthylglucose dans le coeur de rat obèse n'est jamais compensé par d'autres simuli tels que l'augmentation du travail cardiaque ou le glucose. Ce défaut du transport est la cause probable de la diminution du métabolisme de glucose basal et stimulé par l'insuline. La corrélation entre le transport et le métabolisme du glucose montre que le transport est l'étape limitante du métabolisme dans toutes les conditions testées pour le coeur de rat obèse, ce qui n'est pas le cas pour le coeur de rat témoin. De plus, la diminution dela stimulation du transport de glucose chez le rat obèse par le travail cardiaque ou le glucose, suggère que le défaut du métabolisme se situe à une étape postérieure à la liaison de l'insuline avec son recepteur.
La pathologie de l'insulino-résistance s'aggravant en général avec la durée du syndrôme, l'évolution du défaut du transport de glucose chez le rat obèse a été étudiée. Il est ainsi observé que chez les rats
obèses âgés de 5 semaines, le transport de base est encore normal, mais que sa stimulation par l'insuline ou l'augmentation de la pression de perfusion (travail) est déjà altérée. A cet âge, seules des conditions telles que l'adjonction d'insuline associée à une pression· de perfusion plus élevée peuvent normaliser le transport du glucose à des valeurs semblables à celles observées chez le coeur du rat normal testé dans les mêmes conditions.
Dans un troisième temps, les caractéristiques propres des transporteurs du glucose (un protéine spécifique d'un poids moléculaire d'environ 46 kDa) ont été étudiées chez le rat normal en comparant les courbes de liaison de la cytochalasine B aux transporteurs du glucose et celles de la cinétique du transport du 3-0-méthylglucose.
Dans le coeur de rat mince, le glucose seul peut stimuler de quatre fois le transport. De plus, l'insuline double la vitesse maximum de transport de l'hexose et diminue de moitié la valeur du Km.
Finalement la coopérativité entre les transporteurs, calculée selon Hill, est accrue en présence de l'hormone.
Le nombre de transporteurs présents sur la membrane plasmique et dans les microsomes a été étudié par la méthode de la liaison de la cytochalasine B tritiée sur les membranes, deplaça ble par le D-glucose non marqué. Pour mesurer les transporteurs :
a) Les coeurs sont perfusés pendant 15 minutes dans les conditions voulues (insuline, glucose ou 1 et pression de perfusion).
Ceux-ci sont ensuite homogenéisés, puis les membranes plasmiques et microsomales sont préparées par des centrifugations différentielles, notamment sur gradient de Percoll. Ces deux types de membranes ont
été selectionnées car il a précédemment démontré que les transporteurs sont situés dans celles-ci et que l'insuline provoque le mouvement de transporteurs du glucose des microsomes vers la membrane plasmique.
Les membranes sont caractérisées par des enzymes spécifiques, la 5'nucléotidase pour les membranes plasmiques et la NADPH cytochrome c réductase pour les microsomes.
b) Les différentes membranes sont incubées avec plusieurs concentrations de cytochalasine B tritiée en présence de L-glucose pour la liaison totale et de D-glucose pour la liaison non-spécifique. Le D-glucose déplaçant plus de 90% de la cytochalasine B liée au transporteur. La différence entre la liaison totale et non-spécifique donne la liaison spécifique de la cytochalasine B au transporteur de glucose.
Les résultats obtenus avec des coeurs perfusés de rats minces confirment la translocation de transporteurs de membranes intracellulaires à la membrane plasmique. L'insuline double le nombre de transporteurs du glucose présents dans la membrane plasmique, avec une diminution concommitante du nombre de ceux-ci contenus sur les microsomes, sans changer le nombre total de transporteurs. En raison du fait que le transport de 3-0-méthylglucose est accru de huit fois par l'insuline dans les mêmes conditions expérimentales (50 mmHg et 6 mM glucose) , d'autres facteurs que la translocation doivent participer à la stimulation du transport.
En effet, le coefficient de Hill est augmenté par l'insuline, ce qui indique une coopérativité entre les différents sites de liaison spécifiques à la cytochalasine B. Cette conclusion est concevable, car le transporteur existe à l'état de configurations différentes , mono-, di- et
tétra- mériques. Elle est confirmée par la présence d'une curvilinéarité des courbes de cinétique du transport de 3-0-méthylglucose. Dans les microsomes le coefficient de Hill est inchangé par l'hormone.
L'affinité de la cytochalasine B pour le transporteur est également augmentée dans les membrane plasmatiques et microsomales (dans une moindre mesure).
Lorsque les effets de l'insuline sur l'efflux de 3-0-méthylglucose et sur la cytochalasine B sont examinés ensembles, il est possible de conclure que :
a) la translocation est responsable seulement d'une partie de l'augmentation du transport de glucose;
b) la même augmentation a été observée pour l' efflux de 3-0-méthylglucose et pour les transporteurs;
c) l'affinité du 3-0-méthylglucose et de la cytochalasine B pour le transporteur est accrue par l'hormone;
d) l'augmentation du transport de ·3-0-méthylglucose est due à une meilleure efficacité du transporteur du glucose, ce qui est reflet té par l'augmentation de la constante cinétique k (fraction d' hexose transportée par unité de temps).
Ces résultats suggèrent que l'insuline, non seulement promouvait un mouvement de transporteurs, mais peut activer ceux-ci dans la membrane plasmique. La protéine kinase C participe probablement à ce processus, car le transport est stimulable par le phorbol ester, 12-0-tetradecanoyl-13-acetylphorbol (PMA), un effecteur de la protein kinase C et le transporteur de glucose est phosphorylé par cet enzyme.
L'augmentation du travail cardiaque, ainsi que le glucose sont capables de stimuler le transport de glucose par une translocation de transporteurs des membranes intracellulaires vers la membrane plasmique. Ces deux stimulateurs provoquent également une augmentation de l'affinité. Par contre le coefficient de Hill est augmenté par le travail cardiaque, mais pas par le glucose.
Dans le coeur de rats génétiquement obèses (fa/fa), la liaison spécifique de la cytochalasine B a été mesurée pour tenter d'expliquer la diminution du transport basal et stimulé par l'insuline. Le nombre total de transporteurs du glucose est diminué de moitié, avec comme et un abaissement de cinq fois du nombre de ceux-ci dans les membranes plasmiques est mesuré. L'affinité de la cytochalasine B pour le transporteur est également diminuée de moitié dans les membrane plasmique du coeur de rat obèse.
L'effet de l'insuline sur le mouvement de transporteurs des microsomes vers la membrane plasmique est normal si l'on tient compte de la diminution du nombre de transporteur à l'état de basal. L'insuline n'a aucun effet sur l'affinité de la cytochalasine B pour le transporteur.
Cela suggère une absence d'activation additionnelle du transport au
·niveau de la membrane plasmique. Ceci est confirmé par l'absence de
stimulation, chez le rat obèse, du transport par le phorbol ester (PMA).
Le coefficient . de Hill est fortement augmenté pour les transporteurs présents dans la membrane plasmique du coeur de rat obèse. Ce phénomène semble être compensatoire de la diminution du nombre de transporteur ainsi que celle de l'affinité. L'insuline, le
travail ou le glucose décroissent ce coefficient de Hill élevé à des valeures inférieures à celle du rat témoin.
Ce double défaut de la synthèse (ou de la dégradation) du transporteur et de son affinité pour la cytochalasine B explique la moindre capacité du coeur de rat génétiquement obèse à utiliser le D-glucose. L'insuline, le travail cardiaque ou le glucose n'arrivent pas à compenser la diminution de l'utilisation du D-glucose.
L'insuline étant connue pour réguler la synthèse de certains enzymes, il est concevable que l'hormone stimule la synthèse du transporteur de glucose et que cette étape soit insulino-resistante chez l'animal obèse.
L'ensemble de ces données, toutes originales, donne une notion de la régulation de la captation du glucose, tant chez l'animal normal que chez l'obèse. Chez l'obèse insulino-résistant ces résultats montrent (pour le coeur) et suggèrent (pour la masse des muscles squelettiques) la diversité des altérations "post-récepteur", même lorsque l'on se confine à l'étude d'une seule fonction telle le transport du glucose.
I. INTRODUCTION
The present work is composed of two sections
a) The first part describes the state of the art of insulin action on glucose metabolism and transport in peripheral tissues from normal rat. The insulin action on glucose metabolism of insulin-resistant, genetically obese (fa/ fa) is th en discussed.
b) The second part presents the original work, performed by the author, on glucose metabolism and transport in hearts from lean and genetically obese (fa/fa) rats.
1. A. INS ULIN ACTION
I.A.1. Insulin binding toits receptor
In 1975, kinetic studies established th at [ 125
] I insulin-receptor interaction was characterized by a binding constant 0.37 mU/ml and by a curvilinear Scatchard plot (103). Subsequent experiments led to the conclusion that the insulin receptor was principally formed of two subunits, the alpha and ·the beta, of 125 kDa and 90 kDa, respectively.
A heterotetrameric disulfide-linked receptor structure was proposed (38 ,52 ,59 ,82). Incorporation of labelled saccharide into the two subunits demonstrated that both the alpha and the beta subunits contained oligosaccharide units ( 45) . The beta subunit has been shown to be associated with an insulin-stimulated tyrosine kinase activity (60).
This kinase auto-phosphorylates the beta subunit in vitro, suggesting th at the kinase activity could play a regula tory role. There is considerable information about the interactions of insulin with its receptor, as weil as about insulin and metabolic pathways. However, the biochemical-biophysical link between receptor function, subsequent
intracel~ular events and final biological effects is still unresolved. One hypothesis suggests that such a link may involve the receptor-associated tyrosine kinase activity which, directly or indirectly, would modula te other protein kinases and phosphatases, and could be responsible for the final biological effect (17).
One characteristic of insulin action is that its maximal effect is obtained when only a small fraction of the total amount of available receptors are occupied by the hormone. The seemingly "unnecessary"
. receptors are referred to as spare receptors. When considering insulin
glucose, ami no acids or potassium uptake) can all be explained by the existence of a single type of homogeneous receptors, with the different pathways becoming rate limiting at various degrees of receptor occupa ney by the hormone (55). N evertheless, there are spare receptors for ali the pathways just described. The decrease of the amount of available receptor by trypsin allows to show that spare receptors are functionally active (55).
I.A. 2. Mediators of insulin action
When insulin binds to its receptors, a signal(s) is produced that provokes the various biologie al responses. As the mode of action of insulin is still unknown, four major different hypothesis have been proposed for su ch signal: a) protein phosphorylation-dephosphorylation;
b) thiol-redox; c) peptide or phospholipid second messenger; d) protein kinase C.
a) Phosphorylation-dephosphorylation It has become increasingly evident that the rapid effects of insulin on sorne processes may be mediated by changing the degree of phosphorylation of key enzymes of glucose a~d/or lipid metabolism (31)'. In several cases, insulin appears to act by promoting a net dephosphorylation of protein phosphorylated by either cAMP-dependent (e.g. triacylglycerol lipase (92)), or cAMP-in dependent mechanisms (e. g. pyruvate dehydrogenase (32) and glycogen synthetase (97)). However, increased protein phosphorylation in response to insulin has also been reported. This could be the case for the glucose transporter proteins ( 49) and other proteins
(1,8,81,110). These alterations in protein phosphorylation may be due either to changes in the activity of protein kinase(s), of protein phosphatase(s), or to both. The receptor associated tyrosine kinase activity is activated by insulin (139). This bas been the basis for postulating that the receptor tyrosine kinase stimulates a serine kinase which is released into the cytoplasm (39) and which further activates protein kinase(s) or phosphorylase(s).
b) Thiol-redox : Studies on the action of oxidants which mimic insulin effects on fat cells are interesting as they appear to act at a subsequent step to the hormone-receptor interaction (28, 29, 6~). The se studies suggest that oxidation of sulfhydryl group(s) may be necessary for the stimulatory effect of insulin on metabolic pathways.
c) Peptide(s) or phospholipid(s) as second messenger : The concept that insulin regulates the generation of a series of chemical signais from the cell membrane is attractive for two reasons : a) the generation of a chemical 'signal produced by enzymatic events can lead to amplification of a given metabolic process. This concept is weil established in the case of cAMP generation; b) the production of a set of different signais with varying sites of control is in keeping with the multifaceted aspect of insulin action (70). Two different putative mediators have been partly purified from adipocytes and muscle or from isolated membrane plasma membrane of adipocyte. One mediator has been identified as a peptide (71) and the other one as a phospholipid (96).
Both mediators have been shown to modulate the activities of a number of insulin-modulated enzymes (62,63,84,104,105). Further experiments are necessary to elucidate the precise role and nature of the chemical mediator of insulin action.
d) Protein kinase C : In 1977, protein kinase C was identified by Nishizuka as
phosphokinase
a novel (115).
widespread The intact
serine- enzyme
and threonine-directed requires Ca2
+ and phospholipid, in particular phosphatidyl-serine, to express its activity . A wide variety of compounds released during hormone-induced inositol-lipid breakdown have been proposed to act as a second messenger, with protein kinase C as their target (115). In 1982, Castagna et al. (18) reported that protein kinase C can also be activated by "tumour promoters" su ch as 12-0-tetradecanoyl-13-acetylphorbol (usually abbreviated as TPA or PMA). The most active tumour promoters are the most effective activa tors of the protein kinase C. A mechanism by which tumour promoters, or 1,2-diacylglycerol (1,2-DG) stimulate the protein kinase C has been proposed : the intercalation of either 1 ,2-DG (by hormone action or by direct addition of synthetic DG) or a tumour promoter causes a translocation of the protein kinase C from the cytosol to the plasma membrane and activation at the inner surface of the plasma membrane (137). Although the nature of interaction between protein kinase C and insulin action is still undefined, current knowledge suggests that the protein kinase C can modulate insulin action by phosphorylating common targets ( 116,123). The se include the B-subunit of the insulin receptor (116) and possibly the glucose transporter (49,136). The interaction between protein kinase C and insulin action have been confirmed by the observation of insulin-like effects in cells incubated with phorbol esters: inhibition of ketogenesis ( 122), stimulation of lactate production ( 15) , of glucose oxidation and lipogenesis ( 109) , activation of tyrosine aminotransferase, of glycogen synthase (116) and pyruvate dehydrogenase (37).
1. A. 3. Glucose transport
The study of sugar transport started in 1930 by a work carried out on human red blood cells. At that time, the transport was thought of as a non-mediated diffusion, even if a clear deviation from Fick's law
(i.e. non-mediated diffusion) was noted ( 5, 85,95). La ter, saturability and specificity as weil as a number of other features incompatible with non-mediated diffusion have been demonstrated (75). The concept that the transport of glucose across the plasma membrane of skeletal muscles might limit the rate of metabolism was first put forward by Lungsgaard (80) in 1939 who demonstrated that muscle cells contain extremely low levels of free glucose, th us placing the rate limiting step prior to initial phosphorylation of glucose by hexokinase. The role of insulin on the activity of the glucose transport system was shown by Levine et al.
(76) and by Morgan et al. (86) in 1964. Finally, the use of adipocytes by Rodbell in 1964 (102) was an important milestone, as this technique has been widely used for the characterization of the insulin-sensitive hexose transport system.
The plasma membrane is equipped with specifie proteins referred to as "carriers" or "transporters". These proteins greatly facilitate the transfer of glucose from the extracellular space to the cytosol.. In addition, glucose, can enter into a cell by simple physical diffusion, a rather slow process. The rate of hexose equilibrium a cross the plasma membrane is th us mu ch higher by a factor of 10.000 fold than it would be non-mediated diffusion ( 40). Glucose crosses the plasma membrane via two different transport systems dependin g upon the cell type :
- In intestinal and kidney cells, glucose enters the cell against a gradient concentration. This transport is active and is driven by a Na+ gradient(135).
- ln muscle and adipose cells, D-glucose crosses the plasma membrane through specifie trans porters, a process referred to as passive or facilitated transport. Un der the se conditions, the difference of glucose concentrations between both si des of the membrane (i.e. gradient of substrate concentration across the membrane) determines the direction of the passive transport. In contrast to the non-mediated diffusion, the transport system shows saturation curves which can be described kinetically by using equations analogous to those applied in enzymology ( 135) . Although data exist for cell types other than adipocytes ( e. g.
cardiocytes (35)), ail the theory concerning the insulin-sensitive hexose transport was first obtained by experiments carried out on adipocytes.
This explains the use of data taken from experiments on adipocytes to introduce the theory for the method developed in the present work (see II. A.) , which permits the measurement of glucose transport in the perfused heart.
Kinetic description of transport involves the measurement of flux through the membrane. Sin ce D-glucose is rapidly metabolized, it is impossible to study its transport directly. Therefore, transported and non-metabolized su gars such as 3-0-methylglucose (3-0-MG) (26 ,124) have been used. The non-facilitated transport (free diffusion) represents only a negligible part of the overall transport under most of the experimental conditions. This statem-ent is in part derived from the following observations : the equilibration time for L-glucose is more than one hour when the half-time for 3-0-MG entry is 2-3 seconds
( 133); only a small fraction of 3-0-MG diffusion is not inhibited by cytochalasin B, a drug that interferes with the mediated transport of the hexose (124).
The binding of the hexose to the carrier will form a complex which undergoes a transconformation able to dissociate on the opposite leaflet of the membrane. Figure 1 shows the model most widely used to describe the kinetic properties including those of saturation :
Figure 1 Carrier model of glucose transport
c
carriers
substratecs
carrier-substrate complexSo Si
I inside the cell 0 outside the cell
The substrate flux is described by the Michaelis-Menton equation : v= (Vmax
*
S)/Km + S (36). To follow the flux of a non-metabolized substrate from one face of the membrane to the opposite face and to determine its characteristics, Km and V max, four main protocols are used : a) the equilibrium exchange experiments are carried out with an unidirectional flux of radiolabelled substrate, wh en the concentration of unlabelled substrate is equal on both si de of the membrane; b) thezero trans influx experiments consist in following the entry of labelled sugar in the absence of both labelled and unlabelled sugar inside the cells at the initial ti me; c) in the infinite cis influx experiments, the net flux of labelled sugar is measured when the external space is saturated with unlabelled sugar; d) the infinite trans influx experiments measure the net flux of labelled sugar when the cells are saturated with unlabelled sugar. These protocols permit also to follow efflux of labelled sugar.
The kinetic parameters for 3-0-methylglucose transport in the adipocyte of rats are summarized in Table 1.
TABLE 1 Effect of insulin on kinetic parameters, i.e. V max and Km of 3-0-methylglucose transport measured by different experimental protocols in adipocytes
Ex periment Reference Km [mM] Vmax [mM/&]
Basal + insulin basal + insulin
Equilibrium exchan ge Vinten et al.(125) 5 5 0.07-0.2 1.6-1.9 Whitesell et al. ( 133) 2.5-5 2.5-5 0.058 0.8 Taylor et al. (118) 4.22 4.45 0.058 0.84
Whitesell et al. ( 132) 35 3 0.150 0.750
Zero trans entry Whitesell et al. (133) 2.5-5 2.5-5
Taylor et al. (118) 5.41 6.1 0.034 1.2
Zero trans exit Taylor et al.(118) 4.09 2.66 0.153 1.19
Holman et al. ( 48) 5.65
Infinite cis entry Taylor et al. ( 118) 9.03 6.51 0.066 0.98 Infinite cis exit Taylor et al. ( 118) 4.54 3.60 0.106 1.76
Sever al conclusions can be drawn from the se parameters. The symmetry of the transport is the first question that has to be posed (i.e. are the kinetic parameters the sa me for influx and efflux ? ) . This is of primary importance since the method for measuring glucose transport used in our work, assumes a symmetrical glucose transport.
The data presented in Table 1 provide no evidence for an asymmetry of 3-0-methylglucose transport in adipocytes. As can be seen, there is no significant difference between the Km for influx and efflux, respectively ( 48,99,118,125,132). These conclusions are not necessarily representative of all type of cens: the erythrocyte glucose transporter does present a marked asymmetry (134). Thus, on the basis of the studies just summarized, it is possible to identify at least two classes of sodium-independent, facilitated diffusion systems for hexoses in mammalian tissues: those with symmetrical kinetic parameters and those which show asymetrical ones .·
Of importance is to note that insulin does not alter the symmetry of the symmetrical glucose transport (118 ,133). The effect of insulin on the Km and Vmax of the hexose carrier is the second question that should be discussed on the basis of results presented in Table 1.
Insulin augments the maximal transport velocity (Vmax) for both the influx and the efflux of the sugar by a factor of 5 to 35 fold depending on the experiments (21,27 ,48,118,125,132,1.33). The effect of insulin on the Km is more controversial. Un til recently, the Km was claimed to be unaffected by the hormone ( 48,118,125,133), but a recent work of White se li ( 132) demonstrates th at it does modify the Km, the la ter being 35mM in basal state and 3mM in the presence of insulin. The rea son for the insulin-induced increase in Vmax is probably that more
transporters are present in the plasma membrane, as proposed by research groups who have used the following techniques
- cytochalasin B binding to membranes (126) - transport activity in a cell-free system (113) - photoaffinity labelling of the transporter (93)
Cytochalasin B is a fungal substance that inhibits the glucose transport in a variety of cell types (11, 79, 117), in both insulin-insensitive (77, 117) and insulin-sensitive ones (79). The binding of cytochalasin B is highly specifie for the glucose trans porters. It shows a very high affinity for the transport as only a high concentration of unlabelled D-glucose (500mM) can inhibit its binding to transporters at more than 90%, L-glucose being unable to do it ( 126). Furthermore, the cytochalasin B bound to its transporter can be activated by U.V. light and will covalently react with the protein ( 93) .
Cytochalasin B binding to isolated membranes : Wardzala et al. (126) used the [3
H] cytochalasin B binding technique in partially purified plasma and microsomal membranes from fat cells to quantify the number of glucose carriers. The cytochalasin B bound to membranes in presence of L-glucose represents the total binding, which is the binding on glucose transporter plus the non-specifie one. The D-glucose is able to displace cytochalasin B bound to its transporter.
Th en, the fraction of the cytochalasin B bound to membranes incubated with D-glucose (500 mM) represents the non-specifie binding.
Consequent! y, the difference in bindin g in presence of either D-glucose or L-glucose at a given cytochalasin B concentration represents the specifie [ 3
H] cytochalasin B binding to the glucose transporter(see
D-G
CBT + CBM + CB -~---~• + CB + CBM+ T
t
L-G
T
=(CBT + CBM + CB)
L-G -(CB + CBM +
1) D-GCB : Cytochalasin B
CBM : Cytochalasin B bound to membranes CB T : Cytochalasin B bou nd to
transporters T : Transporters
L-G: L-glucose
D-G: D-glucose
Figure 2). The number of glucose transport systems and the dissociation constant (Kd) were calculated from the Scatchard plot ( which was reportedly linear) of four points of specifie [ 3
H]
cytochalasin B binding. Studies of cytocha1Rsin B binding led to the following conclusions : a single class of glucose carriers appears to be present in both plasma and microsomal membranes of normal fat cells.
Insulin increases the number of glucose transporters in the plasma membranes through an apparent stoichiometric translocation from an intracellular pool of carriers located within microsomal membranes (24 ,126). These experiments have been subsequently extended : the action of insulin on translocation from microsomes to plasma membrane is dose dependent (half max. 15 J,JU/ml), time dependent (max.
translocation 5 min) and reversible (58). Furthermore, the comparison between the insulin effect on the 3-0-methylglucose transport and on the number of glucose transporters present in the plasma membrane showed a lag of 1. 5 minutes between the appearance of intracellular glucose transporters in the plasma membrane fraction and the corresponding rise in actual glucose transport ( 43,58, 133). This suggests the existence of at least one additional phenomenon between translocation of glucose transporters to the plasma membrane and actual effect on glucose transport ability.
Transport activity in a cell-free system : Suzuki and Kono performed the reconstitution of 3-0-MG transport in liposomes with proteins extracted from various membrane subfractions (113). The transporter were enriched in fractions of plasma membranes when the cells were previously treated with insulin ( 113).
Photoaffinity labelling of the transporter : The two previous approaches just summarized give an interpretation of the mechanism of insulin action without demonstrating the individual steps involved in the translocation process. One should note th at other possibilities th an the translocation are conceivable. For example, the membranes containing glucose transporters could be part of the cell surface and share microdomains with the plasma membrane. These microdomains could be separated from the plasma membrane by the homogeneization and be fractionnated as microsomes. This phenomenon has been observed in lymphocytes ( 47). Insulin might act by inducing structural change in these membrane microdomains that are no more separated by homogeneization. A technique was developed to locate the intracellular pool of trans porters ( 93). Intact cells or membranes were exposed to
[3
H] cytochalasin B and UV light, the latter producing covalent binding of cytochalasin B to the glucose transport ers. The membranes were then disrupted and proteins were separated by polyacryamide gel electrophoresis ( 68). The results demon strate th at wh en the fat cells had been prelabelled with [3
H] cytochalasin B in the absence of insulin, subsequent addition of the hormone produced the appearance, in the plasma membrane, of glucose transporters previously sequestered in the cell interior . The effect of insulin to increase the translocation of sequestered transporters to the external medium is approximately 4-fold. This confirms the observation of 2 to 5 fold increase of glucose transporters located in the plasma membrane measured in the above described experiments (93, 113, 126). This 2-5 fold stimulation of the translocation could be related with the 5-35 fold increase in sugar transport measured in intact fat cells ( 48,118,125,132,133). Therefore, the translocation process as initiated by insulin of the transporters might be only part of the mechanism by which insulin stimulates glucose
transport. An alterna te way of increasing the glucose transport by insulin could be an augmentation of the affinity of glucose for the transporter. A major unresolved question is th at of the location of the presumed intracellular trans porters. Sin ce the Golgi-enriched or the micro som al fraction, in which the se are found, are a heterogeneous mixture of membranes, it remains to be demonstrated from which specifie (if any) structure, the transporter-containing membranes derives. It may be possible, for example, to ultimately identify their localization by immunofluorescent-electromicroscopy, using an an ti transport er
known. The
antibody once the structure of the transporter is exocytotic-endocytotic mechanism for the reversible modulation of hexose transport may be involved, as glucose transport in diaphragm (130) and in heart (131), other transport systems (87, 129, 138) are regulated by such process.
The Figure 3 summarizes the overall mechanism of hexose transport regulation. The association of insulin with its specifie receptor ( 1) genera tes sorne unidentified signal ( 2) that appears to initia te the recruitment of small intracellular vesicles containing the glucose transporter ( 3). The se vesicles would bi nd to ( 4) and immediately fuse with the plasma membranes (5), thus exposing functional glucose transporters and allowing glucose influx (6). The reversai of this process following dissociation of insulin from its receptor (7) would involve back translocation (8) of vesicles containing glucose transport systems to the cell's interior. Such recycling could also occur as long as receptors are occupied by the hormone. In basal state, the relative rates for the various steps of the recycling process would be such that the steady state distribution of the transporters favors the cytoplasmic localization. Insulin could interact at any of the se steps, however, th at
figure 3 : Schematic representation of a hypothetical mechanism of insulin's stimulatory action on glucose transport in the isolated epididymal adipose cell.(Data from Karnieli et al. 58 )
lntraceflular
p~
at the plasma membrane level will not require an intracellular second messenger. The nature of the respective step(s) responsible for the translocation process re mains to be established.
1. A. 4. Glycolytic enzymes in muscle
The main pathway of glucose metabolism in muscles represented by glycolysis, while pentose cycle and gluconeogenesis have a mi nor or negligible role.
Hexokinase and phosphofructokinase have a relatively low activity compared to the other enzymes of the pathway. This, together with the weil known allosteric properties of the second enzyme, makes these of high regulatory potential. Insulin has no effect on hexokinase activity in vitro in extract of rat muscle (88). On the contrary, phosphofructokinase stimulation by insulin has been suggested to be responsible for the enhancement of the glycolytic rate· produced by the hormone in rat muscle. This effect appeared to be· in dependent of the action on glucose transport (7 ,89). Therefore, hexokinase and phosphofructokinase are both candidates as bèing the rate limiting steps for glycol y sis, at least in he art ( 64,91).
I. B. INSULIN RESISTANCE IN GENETICALLY OBESE (fa/fa) RATS
1. B .1. Syndromes of obesity
Animal models with spontaneous (e. g. genetic) or experimentally produced (e.g. hypothalamic lesions) obesities are numerous (2). The abnormalities reported in these models vary from one animal type to
another, making it unlikely th at the obesity syndrome can be reduced to a single pathophysiologic entity. However, wh ether rats or mice made obese by hypothalamic lesions, or genetic ohe se-hyperglycemie mi ce (double recessive genes : ob 1 ob, db 1 db, rn db 1 rn db types) or obese rats (fa/fa types) are considered, in all of them, similar pathological traits and trends have been observed. Thus, obesity has been. shown to be a progressive pathology that comprises two main ·phases : an earl y one without insulin · resistance, a la ter one in which insulin resistance is apparent (2, 14). Hyperphagia and hyperinsulinemia are often concomitant in animal obesities. Hyperinsulinemia is not only a direct consequence of hyperphagia, sin ce it has been shown that hyperinsulinemia and obesity can exist without concomitant hyperphagia ( 41). Th us, hyperinsulinemia probably represents a major abnormality of both the spontaneous or experimentally-produced animal obesity (53).
Note that sorne of the obesity types present have normal basal glycemia (fa/fa type) while sorne other show basal hyperglycemia (ob/ob type).
The early phase of obesity is the period during which augmentation of fat accretion occurs. The liver and the adipose tissue are the two main tissues participating in such increase in fat depot. lt has been hypothetized that the initial disorder(s) of obesity syndromes may lie somewhere within the brain, as two main series of changes of the autonomie nervous system appear to predominate :
- an increase in efferent parasympathetic pathways, with consequent increased insulin secretion which favor fat accretion (increased liver · and adipose tissue lipogenesis, increased hepatic VLDL output, increased uptake of VLDL by adipose tissue via insulin-increased activity of lipoprotein lipase activity (LPL)).
- a decrease in sympathetic pa th ways which, QY alleviating possible inhibitory influence at the endocrine pancreas, could further favor insulin secretion, and by acting to a lesser extent on brown adipose tissue, diminishes energy expenditure (54) .
Th us, the increase in fat accretion is a likely consequence of the CNS-borne dysfunctions just mentioned. At a later phase of the syndrome (the time of appearance of which is dependent on the species studied), hyperinsulinemia is much more pronounced than during the earl y one. This la ter phase is characterized by a state of insulin resistance (2), which will be defined below (see I.B.2. and I.B.3.).
In vivo, the insulin resistant state can be demonstrated by the observed failure of exogenous~y administered insulin to lower blood sugar (2). In vitro, the resistant state is noticed at the level of skeletal muscle, li ver and adipose tissue.
I. B. 2. In vivo insulin resistance
With regard to glucose handling, the two main actions of insulin in normal animais are the stimulation of overall glucose metabolism and the inhibition of hepatic glucose production (119). Abnormalities of these pathways lead to a number of pathophysiological states, among which insulin resistance is a predominant one. Insulin resistance exists when a given insulin concentration produces less than the normal expected biological effect ( 94) . Insulin resistance is frequent! y observed in obesity. Experiments performed in vivo (e. g. via euglycemic clamps) suggest that in hyperinsulinemic insulin-resistant obese rats, the well-substantiated increase in plasma insulin levels becomes unable to stimula te total glucose metabolism. This defect can be related to
receptors as well as post-receptor defects of peripheral tissues (i.e.
muscles mostly) ( 120). Similar conclusions have be en reached for patients with hyperinsulinemia and insulin resistance ( 107). Another major defect observed in vivo is the inability of even very high insulin levels to shut off hepatic glucose production (120). To better understand the defects causing insulin resistance of peripheral tissues, (the main goal of our persona! work), those were also investigated in vitro.
I. B. 3. In vitro insulin resistance of muscles
It was initially thought that the major cause of insulin resistance in obese hyperinsulinemic animais was the decreased ability of liver (57, 78,111) , adipose tissue (94) and muscle (22,72) to bind insulin, an abnormality that could be accounted for by a decrease in· specifie insulin receptor number (56). However, the observation that the decrease in receptor number never required the use of all the spare receptors ( see I. A .1.) of muscle ( 22) ruled out a crucial role of decreased number of receptors in the diminished glucose metabolism of muscle from obese animais. Subsequent studies have indeed suggested th at additional defects, unrelated to the binding of insulin to its receptor (i.e. post-binding defects), contributed to insulin resistance (2,25,30,74a). In insulin-resistant muscles, several such post-binding defects have been described (9,22,23,30,61,72,73,74a). In isolated soleus muscles of the genetically obese (fa/fa) rat or goldthioglucose treated mouse (hypothalamic obesity), the following post-binding abnormalities have been shawn a decrease in receptor autophosphorylation ( 7 4a) , an increased utilization of endogenous fa tt y acids inhibitory to glycolysis (22) and a decreased uptake of a
D-glucose analog, 2-deoxy-D-glucose (2DG) (22 ,23, 72). In perfused hindquarter of genetically obese hyperglycemie (db 1 db) mi ce the existence of a post receptor defect at the lev el of glucose, measured with 2DG, has been proposed to be the major cause of insulin resistance (19). Th us, the evidence that insulin resistance could be partly attributed to defective glucose uptake and metabolism is indirect, based either on overall glucose metabolism or on 2DG uptake. The se facts were taken into consideration when we choose to study the problem of insulin resistance in relation to the glucose transport.
The signal from the insulin receptors, consequent to insulin binding, and the regulation of the glucose transport have not been investigated in obesity, although alteration of those steps could be important in a defective glucose metabolism. The pentose cycle and gluconeogenesis have a minor . or negligible role in insulin resistant muscles. In the obese mouse, hexokinase activity of muscle was found to be unchanged ( 108) , suggesting th at phosphofructokinase is the most probable rate li mi ting step of glycolysis ( see I.A. 4.). As phosphofructokinase has been shown to be stimulated by insulin, an altered stimulation of this enzyme by the hormone is a likely cause of a decreased glycolytic flux. Unfortunately, the re is to our knowledge no information on su ch defect. Fin ally, an inhibition of key glycolytic enzymes in muscle may result from increased levels of free fatty acids (FFA), according to the theory of glucose-fatty acid cycle (100). Fatty acids and ketone bodies would act following their oxidation to acetyl-CoA. This does result in increased formation of citrate, which inhibits glycolysis at the level of phosphofructokinase in presence of ATP. Inhibition of phosphofructokinase causes an increase in the concentration of fructose-6-phosphate; bec a use of the equilibrium
reaction catalyzed by glucose phosphate isomerase, the concentration of glucose-6-phosphate will also in crea se, and will inhibit hexokinase (101, 121) with the final consequence of a decreased glucose uptake.
II. EXPERIMENTAL : GLUCOSE METABOLISM AND TRANSPORT IN HEART AND GENETICALLY OBESE (fa/fa RATS
The present experiments were undertaken to assess the existence , in heart of genetically obese (fa/fa) rats, of possible defects in glucose metabolism that could give insights as to the underlying mechanisms of state of insulin resistance. In the first part of the study, the global glucose metabolism was studied. Subsequently, possible defects of the glucose transport per se were investigated and, finally, alterations of the regulation of glucose trans porters were assessed. In addition, the normal regulation of the glucose transport was further examined, in lean rats, as the translocation process alone · cannot account for the total stimulation of glucose transport in the presence of insulin. The muscle tissue is considered as the most important one in the overall homeostasis of glucose metabolism. Therefore, an alteration of the glucose metabolism at this leve! could be partly responsible for the occurence of glucose intolerance observed in the whole organism.
The choice of heart as a model was made on the following basis a) skeletal muscle such as soleus are using 70-80 % of the needed substrates as lipids (22), making it difficult to investigate glucose uptake; b) in contrast, heart when perfused at 50 mm Hg and 6 mM glucose, utilizes more than 90% is used as energy supply (101,114).
The study of certain aspects of insulin resistance presented in this thesis was carried out on genetically obese (fa/fa) rats, whose specifie characteristics are briefly summarized below. The Zucker obese rat (fa/fa) was first described in 1961 by Zucker and Zucker (140). The obesity of the fatty Zucker rat is due to a single recessive gene, fa
(140). The onset of obesity takes place at about 4 weeks of age, although increased fat pad weight can be detected as early as 7 days of age ( 12). The Zucker obese rat is hyperphagic, hyperinsulinemic, but has normal basal blood glucose and glucagon concentration. When the obesity syndrome is well established, the genetically obese rat becomes markedly glucose intolerant. Insulin, proinsulin and glucagon of this rat have similar immunoreactive binding and biological properties than hormones from normal rat ( 67) . Food restriction does not prevent the development of hyperinsulinemia and fat accretion (51).
II.A. MATERIAL AND METHODS
a) Animais : Genetically obese (fa"tfa) rats and their lean control (FA/fa), FA/FA) bred in our laboratories were used at 5 or 15 weeks of age. They had free access to a standard la bora tory chow ( UAR, Epinay/Orge, France) and were maintained at a constant temperature (23°C) in an animal quarter, with a 12 hour artificial light cycle.
b) He art perfusion Rats were anesthetized with pentobarbital (90mg/kg). The heart was rapidly removed, placed in ice-cold 0.9 % Na Cl for 10 seconds, cannulated via the aorta and perfused, according to the Langendorff technique (90, 114) in which the perfusion medium is infused into the left ventricle via the aorta just above the coronary arteries, as shown by Figure 4. This apparatus allowed two kind of perfusions : a) a "flow-through" perfusion, in which the medium passes only once through the heart, to th en be discarded or collected; b) a recirculating perfusion, in which the same buffer passes several times through the he art. The perfusion medium consisted of a Krebs Ringer
system using another given medium (e.g.medium 1).
TRAP
MEDIUM
n OUT LET
Figure 5 : Experimental design of heart perfusion used for measurement of total glucose metabolism or lactate+pyruvate output. Hearts were preperfused during 3 minutes, then perfused for 10 minutes with a recirculating medium. A first sample of medium was taken at minute 3 and a second one at minute 13, for substrate measurements. A similar design was used to measure glucose metabolism intermediates or the state of the "glucose transport system". ln the case of glucose metabolism intermediate and transporters
measurements, hearts were freeze-clamped in liquid Ni at minute 13.
0 . 3
surgical preperfusion procedure (flow- through)
~me
tample 1
G
perfusion (recirculation)
eample n 13 minutes
clamp
bicarbonate buffer at pH 7. 4. For measurement of glucose metabolism, and the nu rn ber of glucose transporters, a 6 mM physiological concentration of glucose were selected. In studies of 3-0-MG transport, glucose was replaced by 5 mM pyruvate as energy source (22).
c) Glucose metabolism : To measure total glucose meta'Qolism, hearts were pre-perfused for 3 minutes using the "flow-through" technique.
Th en, hearts were perfused for 3 additional minutes with a recirculating medium. After this a first sample of medium was taken. A second sample was collected 7 minutes la ter. This experimental design is depicted by Figure 5. The concentration of glucose was measured in each sample by a glucose analyzer (Beckman, Fullerton, USA) whereas lactate (10) and pyruvate (10) were measured fluorimetrically. The
glycolysis was determined as the production of [3
H]OH from 5-[ Hl glucose (23). The overall glucose metabolism as weil as lactate 3
and pyruvate production were measured as the differences of substrate concentration in the perfusion medium between the two sampling.
Glucose disappearance, lactate production and glycolysis were linear for at least for 25 minutes, as shown in Figure 6. At the end of the perfusion, he arts were freeze-clamped with Wollen berger pinces cooled in liquid nitrogen, for subsequent measurements of metabolic intermediates, dry weight and membrane preparations. Frozen hearts were homogeneized (glass homogeneizer, ABS, Geneva, Switzerland) in 5 % HCl0
4 (w/v) and centrifuged at 2000 g for 10 minutes.
Supernatants were neutralized with K
2
co
3 for fluorimetric measurements of citrate (98), fructose-6-phosphate (69), and glucose-S·phosphate (69). The de novo synthesis of glycogen was measured by the incorporation of 5-'[ H]-glucose (0.5 3 ~Ci/ml) into glycogen (23). The glycolysis was assayed by triated water production from 5-[ Hl-glucose 3(bottom panel) in hearts of normal rats. The three different parameters are measured in hearts perfused at 50 mm Hg pressure, with 6 mM glucose and 10 rn U /ml insu lin using a Krebs-Ringer bicarbonate buffer. Ail the points are in absolute concentration and are the mean ± SEM of 3 experiments.
a
130
-~-~
120
~-2--
""'
c,
~
~"i-
'
e QI 110~f--_____
...
•
100..
0u
~
~ 0 90
80
0 3 6 9 12 16 18 21 24 27
b Tlm• (min)
1.5
rl"v1
...
k
~
QI 1.0
(
.'
!
0
E
;r
... E
... •
0.5
/~
...
..
u
..
/Q....
0
/9.
0 3 6 9 12 15 18 21 24 27
Tl me (min)
c
60
... 40
__--1
lt
~ QI
' ~2
! 30
0
E
?l/2
-=
~ 20
..
llo.. ~0 u
~ 10
0
0
0 5 10 16 20 26
Tl me rmtnl
(50 uCi/ml) (23). Ali the results were expressed by gram dry heart weight ( 101).
d) Rate of 3-0-methylglucose efflux : To measure glucose transport per se, he arts were pre-perfused for 3 minutes using the flow-through
- - -
technique mentioned above and schematized by Figure 7. Subsequently, they were perfused, in absence or in presence of various insulin concentration, with a medium containing 6 mM [ 14
c] 3-0-methylglucose (3-0-MG) 0.3 uCi/ml and 6 mM [3
H]L-glucose (L-G) 0.3 uCi/ml (labelled L-glucose was used to measure the extracellular and free diffusion components). Ten minutes were necessary to achieve the equilibrium. At the end of the loading period, perfusion was continued without re circulation with a medium devoid of sugar. Samples of the medium were collected every 12 seconds in a fraction collector (Gilson TDC 2·20, V illier le Bel, France). 3-0-MG is in the intracellular and extracellular space, L-G being mainly in the extracellular space and to sorne extent, via simple diffusion, in the intracellular spa ce. The. 3-0-MG efflux could be calculated by subtracting for each vial, labelled L-G content from labelled · 3-0-MG content. The plot of cumulative effluxes as function of ti me permitted to deduce kinetic parameters of efflux.
Various tests of linearity (e.g. 0, 1, 2 order kinetics) indicated that cumulative efflux curves fitted the first order kinetic formula : A(t) = A (l-e -kt), where A(t) is the amount of effluent hexose(s) at a given
0
time t, A the maximal quantity of effluent hexose at infinite time or
0
the quantity present in the heart at the end of the loading period, e is the basis of natural logarithm, the kinetic constant k is the fraction of sugar released per unit of time. The rate of efflux (v) during a small period of time was v = dA/ dt = kA kt. To mimic glucose transport, the
0
rate of 3-0-MG efflux was calculated with a t value close to 0 second.
marker) until equilibrium was reached. The efflux was measured by switching to a medium containing only Krebs- Ring er bicarbonate and by collecting the medium every 12s. with a fraction collector. The amount of 3-0-MG transported was equal to the total amount of 3-0-MG minus the LG present in each fraction. (Lower panel) Experimental design for the measurement of 3-0-methylglucose transport in perfused heart.
LOAOING
KRB
L·Gic 3~G
L·Gic 3-Q-MG--+
E XTRACELL INTR~CELL
EQUILI BRIUM
L-Glc
3-Q-MG ~:;; 3-0.MG
ElT. IHT.
E FFLUX KRB
L-Gic
3-Q-MG._- 3-Q-MG
EXT. IHT.
i
lJ H / l.:f
L-Gic ( 3·0-MG tot~l) (tot~llFRACTION COLLECTOR
C~ul.ition 3-Q-MG tr~nsporttd • tot~l 3.()-MG minus tot~l L-Gic
0 3
surgie~ prt~rfusion lo~ding with ~~~led 3-Q-MG
proctdur« ( ftow through) ~nd L-G ( 6mM) (rccircul41tion)
10 15 minutes
l_
fr««ZC Clèmp coll«ction of ctflux«very 12 sec.
(flow through)
The initial speed (V.) was thus V.
=
kA expressed as J,Jmole/min/g dry1 1 0
weight. Wh en the effect of insulin was studied, the hormone was added during both preperfusion and the perfusion periods. When basal glucose or 3-0-MG efflux were investigated in the absence of insulin, the pre-perfusion period was 7 minutes sufficient to wash away preexisting insulin from the hearts.
e) Plasma and microsomal membrane of heart : Hearts were perfused during 15 minutes in presence or in absence of insulin and at a physiological concentration of glucose in the medium (6mM). Hearts were then disconnected from the perfusion apparatus and immediately frozen in liquid nitrogen. Frozen hearts were stored at minus 70°C. For the preparation of membranes, hearts were transferred into liquid nitrogen and powdered within a cooled mort ar. The powder obtained was homogeneized in 4 ml of buffer A (10 mM NaHC0
3,5 mM NaN
3, pH 7.0) using a glass homogeneizer (B. Braun, Molsungen, FRG) whose rod was connected to a homogeneizer (Heidolph, Model R2Rl, · FRG). Fifty J,Jl of homogenate were used for protein and enzyme marker measurements.
The homogenate was centrifuged for 20 minutes at 7'000 x g in a Sorvall RC2-B centrifuge (Norwalk, USA). The resulting pellet (Pl) was kept for the preparation of plasma membrane, whereas the supernatant (SI) permitted to ob tain purified microsomal membranes.
The first pellet Pl was resuspended with a seringue in 2 ml of buffer B (10 mM Tris HCl, pH 7 .4) and centrifuged for 20 minutes at 1000 x g in a Sorvall centrifuge. The supernatant was spun in a Sorvall centrifuge for 20 minutes at 48'000 x g. The pellet thus obtained was resuspended in 1 ml of the medium B and placed on a 20 % Percoll (Pharmacia, Uppsala, Sweden) solution in buffer C (255 mM sucrose, 10
