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
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.
(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
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
The initial speed (V.) was thus V.
=
kA expressed as J,Jmole/min/g dry 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 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
mM tris-HCI, 2 mM EGT A), centrifuged for 1 hour in an ultracentrifuge (Beckman L5-50, Fullerton, USA) at 55'000 x g using a SW27 rotor . An opaque band corresponding to a density of 1. 030, as measured with density beads markers (Pharmacia, Uppsala, Sweden) was removed. The plasma membranes thus obtained were washed in medium B (see above) and centrifuged for 1 hour in an ultracentrifuge at 170'000 x g, using a 50 Ti rotor (pelleted plasma membranes).
The supernatant SI was centrifuged for 20 minutes at 48'000 x g. The resulting supernatant was placed on top of a 20 % Percoll solution and centrifuged for 1 hour at 55'000 x g in an ultracentrifuge with a SW27 rotor. He re, a band, corresponding to a density of 1. 036, as measured with density beads markers, was collected. The microsomal membranes thus obtained were washed with medium B (as above), and centrifuged for 1 hour at 170'000 x g in an ultracentrifuge using a 50 Ti rotor
(pelleted microsomal membranes).
The pellets of both types of membranes were resuspended in 1 ml of buffer C, of which 0. 7 ml was kept for cytochalasin B binding assays.
The remaining 0. 3 ml was saved for the measurement of enzymes marker and total proteins.
f) Enzyme marker assays Total proteins were measured by the Bio-Rad method, with gamma globulin as standard (13). The 5 'nucleotidase was the marker used for plasma membranes and was determined as described elsewhere (3). The NADPH cytochrome c reductase, a marker of microsomal membranes, was determined as reported before (112).
g) Cytochalasin B binding assays : A cytochalasin B binding curve of zinc sulfate. This precipitated ali proteins, including the glucose transport ers. This particular alteration of the initial method ( 42) was of great importance, as glucose transporters could not ali be pelleted, even after 1 hour of ultracentrifugation at 436'000 x g in a TLlOO centrifuge (Beckman, Fullerton, USA). Indeed, about 25 percent protein was always measured in the supernatent following the ultracentrifugation just mentionned (data not shown). The consequence of the partial and potentially selective sedimention of proteins when using an ultracentrifugation procedure was an underestimation of the cytochalasin B binding. Wh en mentionned, the measured specifie cytochalasin B-binding activities were expressed as pmoles per mg o.f protein and were adjusted to those which would have been observed, had the membrane fractions been free of cross-contamination. Su ch adjustments were based on specifie activities of the enzyme markers and protein recoveries, with the assumption th at 5 'nucleotidase activity is localized specifically in plasma membranes and that NADPH cytochrome c reductase activity is specifie for microsomal membrane (24).
The Hill equation ( 46) was used to analyze the data. The plot of linearized Hill equation, log (b/R
0-b) = n log (F) - lqg(K') was used, in which R
=
maximal binding capacity; b=
bound cytochalasin B; F=
0
free cytochalasin B; n
=
Hill coefficient; K'=
constant comprising interaction factor and intrinsic dissociation constant; Kd = K,lln =dissociation constant) permitted to obtain the Hill coefficient n, the R
. 0
value (maximal binding) and normalized Kd values. The R value was
0
found by a program that iterates calculations with increasing Ro values un til the maximal coefficient of correlation was reached; n was the slope of the linearized Hill equation and Kd = lOlog(F) where
lo~(b/R
-b) =0
0. Using the values of n, Kd and R , the Hill curve was plotted
0
(bound versus free) together with the averaged data points with standard error of the mean of independant experiments.
h) Statistics and calculations Calculations were made on HP 97 (Hewlett-Packard, USA), Cyber 180-830 computer (CDC, USA) and on M24 microcomputer (Olivetti, Italy). Statistical analysis were performed with the one tailed or two tailed Student's t test for unpaired data. The P value indicates the probability th at two events are similar.
II. B. RESULTS
II. B .1. Glucose metabolism
a) Basal and insulin-stimulated glucose metabolism in hearts from 15 week-old lean and obese rats
The insulin dose response curve of glucose metabolism (representing mostly glycolysis) is shawn by Figure 8. In normal hearts, total glucose uptake and metabolism measured by glucose disappearance from the medium reached maximal values at a high insulin concentration (10 mU/ml), half-maximal response being obtained at 5.5 rn U 1 ml. As can also be seen in Figure 8 and wh en expressed in ab solute values, basal and insulin-stimulated glucose metabolism was significantly lower in hearts of obese than in control rats. In particular, basal glucose metabolism was four times lower in hearts from obese rats th an in controls. Wh en the sa me results were expressed as increases of insulin-stimulated over baseline values, both the sensitivity (half maximal effect of the hormone) and the responsiveness (maximal effect of the hormone at maximal concentration) were similar in hearts of lean and obese rats, as shawn by Figure 9. This indicated that the major defect of glucose handling by heart from obese rats is at the lev el of basal glucose metabolism. The low sensitivity of perfused heart to insulin was not due to degradation of insulin : the concentration of the hormone in the medium before and after passage through the heart was measured and no significant change was detected (data not shown).
One hypothesis concerning the low sensitivity of insulin in heart was
E
V)weeks old lean (o..,o) and obese (o-o) rats. The data shown are those of Figure 5 once
that insulin could increase the glucose metabolism by the enlargement of the capillary bed (34). The resulting augmentation in the number of muscle fi bers with glucose could exp lain the increase of its metabolism.
The binding of the hormone on the capillary has been reported, rather th an on cardiocytes, and will further emphasize this hypothesis ( 6) , with the concequence of the intracellular and extracellular space of 3-0-MG increased by the action of the hormone. To answer this question, hearts were perfused in presence of 6 mM 3-0-methylglucose (3-0-MG) and 6 mM L-glucose (L-G). Table 2 shows the effects of insulin on 3-0-MG and L-G spaces in the he art. The intracellular amount of 3-0-MG was increased by insulin from 5 to 19 J,lmole/ gram dry weight (g dw) (nearly 4 fold), whereas the extracellular L-glucose went from 9.1 to 18.6 J,lmole 1 g dw. The re fore one of the action of insulin would be the enlargment of the capillary bed.
b) Effect of endogenous and exogenous fa tt y acids on glucose metabolism of 15 weeks old lean and obese rats
Previous studies carried out in skeletal muscle (the soleus) of obese rodents have shown an increased fatty acid utilization, which via citrate accumulation, inhibits glycolysis at the level of phosphofructokinase resulting in glucose-6-phosphate and fructose-6-phosphate accumulation (22). To search for a similar inhibition occuring in heart of obese rats and thereby be responsible for the absolute decreases in basal and insulin-stimulated glucose metabolism, intracellular concentrations of glucose-6-phosphate, fructose-6-phosphate and citrate were measured. Figure 10 shows that under basal conditions (no hormone) and in insulin-stimulated state (10
2
Figure 10 : Citrate~ glucose-6-phosphate and fructose-6-phosphate content of perfused hearts obtained from 15 weeks old lean (open bars) and obese (fa/ fa) (hashed bars) rats~ glucose metabolism, the values of obese are not statistically different compared to lean;
lactate+pyruvate, the values of obese are not statistically different compared to lean.
4
o_j
Total glucose
metabolism Lactate
+
pyruvate' •
mU /ml) no significant differences between lean and obese rats were found re garding to the respective intracellular concentrations of citrate, glucose-6-phosphate and fructose-6-phosphate. This indicates th at in obese rat, glucose metabolism is not inhibited by endogenous fatty acids utilization. The higher level of citrate observed in presence of insulin (10 mU /ml) in both types of rats must be due to the increase of glucose metabolism. Glycolysis (assessed via [3
H]OH formation from 3
H glucose and via lactate and pyruvate production) is also known to be altered by an over-oxidation of endogenous fatty acids. Additional experiments were carried out to determine whether an exogenous substrate such as acetate would change glucose metabolism (121). lt was found (Fig. 11) that total glucose metabolism was slightly diminished by acetate in hearts from obese rats. However, lactate plus pyruvate production were not different in lean and obese rats. These results depicted by
bars) and genetically obese (Wâ)(hatched bars) rats. When present (+), insulin was at a
d) Glycolysis as influenced by working load
The data of Figure 8 shown above were suggestive of an overall decrease in insulin-stimulated glycolysis in hearts of genetically obese rats. To assess whether this defect in glucose metabolism was due to altered glucose pathway or an abnormality of insulin action per se, the effect of workload was measured since, as discussed below, increasing workload bas an insulin-like effect. In the experiments depicted by Figure 13 workload was defined as the volume of fluid pumped against the impedance of the vascular bed. In the Langendorff method of heart perfusion that we used, a means to increase workload was to augment the perfusion pressure from 50 mmHg to 100 mm Hg, in the absence of insulin. As can be seen by Figure 13, at increased workload, a stimulation of total glucose metabolism of normal rats to the same extent as 10 mU /ml insulin was observed. Total glucose metabolism in hearts from the obese rat was not significantly · lower than lean control.
However, glycol y sis was significantly decreased in heart from genetically obese (fa/fa) rats when compared to controls (Figure 13, lower panel). The main point of these results was to show that glucose metabolism was stimulated to the same extent by either maximal concentration of insulin alone and by a higher working load without insulin.
To summarize at this point, insulin-stimulated glucose metabolism per se was little altered in heart from obese animais. The main finding (shown by Figure 8) was the demonstration of an altered basal glucose metabolism, suggesting the existence of a defect principally at the level of the glucose transport. For this reason emphasis was placed on glucose transport and subsequently on the glucose transporter, in hearts from both lean and genetically obese rats.
II. B. 2. Glucose transport
a) Basal and insulin-stimulated 3-0-methyl glucose efflux in heart of 15 weeks old lean and obese rats
As decreased basal ( without insulin) glucose metabolism appeared to be the major defect of heart from obese rats ( see II. B .1.) , actual 3-0-MG transport was assessed by measuring efflux rates as described in Materials and Methods (II. A.). In normal rats (Figure 14) insulin stimulated 3-0-MG efflux by fourfold when compared to basal values, reaching plateau values within 3 minutes. The half time of the efflux was 36 seconds for basal values and 18 seconds for insulin-stimulated 3-0-MG efflux rates. Of further importance was the observation that insulin-stimulated 3-0-MG efflux process was completely abolished by superimposed addition of cytochalasin B to perfused he arts. As the physiological direction for the D-glucose is influx and the 3-0-MG transport was measured as efflux, the symmetry of the glucose transporter in the heart muscle has to be demonstrated to assess a physiological role to the 3-0-MG efflux. The symmetry of 3-0-MG transport was tested as follows : the heart was loaded with labelled 3-0-MG and L-G (at identical concentration to those used for efflux experiments), and freeze-clamped in liquid nitrogen at various time intervals. Each sample was digested and counted. The 3-0-MG specifically transported was the difference between the content of heart in 3-0-MG and L-G. Identical time course of 3-0-MG influx and efflux showed the existence of a symmetrical transport in heart muscle (Figure 14).
(.!)
~ 0
1 (")
Figure 14 : Cumulative 3-0-methylglucose influx ( ) and: efflux in perfused hearts of 15 weeks-old lean rats in the absence (o-o) or the presence
<•-•>
of insulin (10 mU/ml) or in thepresence of insulin plus cytochalasin B (100 uM) (D-D). The data shown are taken from a representative experiment repeated at least four times.
5
·-·-·-· •
--~-·-4
~
-
t . /res 1 /
~
3
.s: A/
.._
•
0
E 2 . 1
-
:J. o-o-o--<>-0
-o-o--0-~_.o--o
D""'
0
o--0--o-o-a-o--o0 2 3
Ti
me (min)As shown in Figure 15A, basal and insulin-stimulated 3-0-MG efflux by heart of obese rats was significantly lower than that of controls. Upon ta king into account the observation that basal 3-0-MG efflux was decreased in hearts of obese rats (i.e. wh en results obtained with insulin were expressed as increases over basal values) it was found that the sensitivity (half maximal response to the hormone) of heart from obese rats was similar in both group of animais but that there was an impairment in the maximal response to insulin in obese animal (Figure 15B). To access whether the decreased glucose transport (Figure 15) is the cause of the diminished glucose metabolism (Figure 8) , a correlation between tho se two process was performed, as shown by Figure 16. The ratio of glucose transport to glucose metabolism should be 1 if glucose transport were to represent the rate limiting step of glucose metabolism. It appears likely th at glucose transport is rate limiting in hearts of obese animais at ali insulin concentrations used, while it would not be the case for normal con trois. In control,. at two higher insulin concentration, a step subsequent to the transport appears to be rate limiting. The hexokinase is likely that limiting enzyme.
b) Specificity of 3-0-MG efflux
As the transport of 3-0-MG glucose is symmetrical, the presence of D-glucose or another hexose should, wh en measuring 3-0-MG efflux, increase the rate constant k of 3-0-MG efflux {an index of transporter efficiency), and therefore augment the initial 3-0-MG efflux rate (vi=kAo), as function of the potency of the agonist selected. To test this hypothesis he arts were loaded with labelled ( 1 mM final conc.)
Figure 15 : Insulin dose response curve of 3-0-methylglucose efflux in perfused heart of
Figure 16 : Correlation between glucose metabolism and glucose transport in perfused
3-0-MG and L-G, the efflux of 3-0-MG was th en performed with a perfusion medium devoid of addèd substrate or 6 mM fructose or 6 mM D-glucose. The results obtained are shown in Figure 17. The transport was not stimulated by the addition of fructose in the extracellular space when compared with the perfusion medium in absence of added substrate. However, D-glucose markedly increased (7 times) the rate of 3-0-MG efflux in similar experiments (Figure 17C, lower wh en compared with perfusions without substrate, whereas D-fructose . did not modify this constant. The other parameter, A , was unchanged
0
in the three experimental conditions, basal perfusion medium (no additional suga'r present) medium containing D-fructose or D-glucose as illustrated by Figure 17 C . This experiment clearly shows the specificity of the transporter system for D-glucose and 3-0-MG, at the exclusion of th at of D-fructose.
c) Basal; insulin-, and pressure-stimulated 3-0-methyl glucose efflux in heart of 5 and 15 weeks old lean and obese rats
As glucose transport appeared to be the major defect in hearts of obese rats and as the pathology of insulin resistant-animais often changes with the extented period of the syndrome (22), 5 weeks and 15 weeks old rats were compared. As the working load is known to be a
total amount of 3-0-methylglucose contained into the heart. There are not statistical difference between the three groups. The middle panel b) refers to the kinetic constant k (fraction of 3-0-MG transported per unit of time). The difference between control or fructose stimulated and glucose stimulated kinetic constant is statistically significant at P<0.05 by Student's t-test. The bottom panel c) represents the transport of
3-0-methylglucose. Differences between control or fructose stimulated transport and glucose are statistically significant (P<0.002). Each bar is a mean ± SEM of three experiments.
Respective concentrations were D-fructose and D-glucose 6mM; labelled 3-0-MG :
Respective concentrations were D-fructose and D-glucose 6mM; labelled 3-0-MG :