1Department of Sports Medicine, Third Faculty of Medicine, Charles University, 2Diabetes Center, Institute for Clinical and Experimental Medicine, Prague, Czech Republic; 3Obesity Research Unit, Institut National de la Santé et de la Recherche Médicale, Unité 586, and 4Department of Medical and Clinical Pharmacology, Faculty of Medicine, 31073 Toulouse, France
Submitted 14 November 2002 ; accepted in final form 21 May 2003
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ABSTRACT |
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microdialysis; glycerol; isoproterenol; blood flow; 2-adrenergic receptor antagonist
Adipose tissue is also a major target for insulin action. Unlike
catecholamines, insulin inhibits lipolysis and counteracts the action of
-AR by activating phosphodiesterase 3B, which hydrolyses cAMP
(22,
25,
27), and by desensitizing the
-adrenergic responsiveness
(3). Microdialysis allows the
in situ study of the interaction of insulin with adrenergic pathways in
adipose tissue. Catecholamines can be delivered through microdialysis probes
and induce lipolysis stimulation in adipose tissue with no effect on the
circulating concentration of the catecholamines. Simultaneously, insulin can
be delivered intravenously during a hyperinsulinemic-euglycemic clamp with no
changes of catecholamine concentration in plasma or in dialysate from adipose
tissue or muscle (23,
26). Animal studies have shown
a decrease of the lipolytic response to norepinephrine during a
hyperinsulinemic-euglycemic clamp in adipose tissue
(8). From additional human
investigations, it has been concluded that the
-adrenergic lipolytic
effect in adipose tissue is counteracted by the action of insulin
(14,
18). Until now, no study has
taken into account the role of the antilipolytic
2-adrenergic pathway in the in situ adrenergic response when
plasma insulin levels are increased.
The first objective of the present study on the basis of in situ
microdialysis use was to investigate the counteraction of hyperinsulinemia on
the lipolytic effect of isoproterenol (a selective -AR agonist) or
epinephrine (a catecholamine exhibiting both
2- and
-AR agonist properties) in adipose tissue. The second objective was to
delineate the contribution of the antilipolytic
2-AR pathway
to the blunted adrenergic lipolytic response in adipose tissue during
hyperinsulinemic-euglycemic clamp.
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MATERIALS AND METHODS |
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For the study, eight healthy young men aged 23.9 ± 0.9 yr (range: 22-25 yr) with a body mass index ranging from 21.4 to 27.0 (mean ± SD: 23.8 ± 1.9), who had not been submitted to any pharmacological or nutritional protocol before the study, were recruited. All had a stable weight during the previous 3 mo. Selection of the subjects was based on a screening evaluation of detailed medical history, a physical examination, and several blood chemistry analyses. The Ethical Committee of Third Faculty of Medicine, Charles University, approved the study. All subjects gave their informed consent for the experimental conditions after detailed explanation. The investigations were carried out in the Diabetes Center, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
Experimental Protocol
Studies were performed in random order, and the eight subjects were studied on three separate occasions (twice with a clamp and once without). These three examinations were separated by a 5- to 8-day period, and patients were instructed to keep their nutritional and physical activity habits during that time. The subjects entered the hospital at 8:00 AM and were maintained in the supine position during the experimental period. An indwelling polyethylene catheter was inserted into the antecubital vein of each arm. At 8:30 AM, microdialysis probes (Carnegie Medicine, Stockholm, Sweden) of 20 x 0.5 mm and 20,000-MW cutoff were inserted percutaneously into the abdominal SCAAT after epidermal anesthesia (200 µl of 1% lidocaine; Roger-Bellon, Neuilly-s-Seine, France). When two probes were used, they were separated by at least 10 cm and inserted at a distance of 10 cm to the right of the umbilicus. When necessary, a third control probe was inserted in the contralateral side at a similar distance from umbilicus. The probes were connected to a microperfusion pump (Harvard Apparatus, SARL, Les Ulis, France) and infused with Ringer solution (in mM: 139 sodium, 2.7 potassium, 0.9 calcium, 140.5 chloride, 2.4 bicarbonate, and 5.6 glucose). Ethanol was added to the perfusate to estimate changes in the blood flow, as previously described (12, 13). The ethanol ratio was calculated as ethanol ratio (%) = (ethanol concentration in outgoing dialysate/ethanol concentration in ingoing perfusate) x 100. The variations of the ethanol ratio were taken as an index of variations of adipose tissue blood flow (ATBF). After a 30-min equilibration period, a 30-min fraction of dialysate was collected at a flow rate of 0.5 µl/min. Then, the perfusion flow rate was set at 2.5 µl/min for the remaining experimental period. This simplified but relevant and less time-consuming method was selected in this long-lasting study (24, 30). The estimated extracellular glycerol concentration (EGC) was calculated by plotting (after log transformation) the concentration of glycerol in the dialysate measured at 0.5 and 2.5 µl/min against the perfusion rates. The EGC found in the present study fits with previous determinations performed in lean subjects (16, 17). The average recovery of probes was calculated as the ratio of the concentratiom of glycerol in dialysate at the perfusion rate of 2.5µl/min and the calculated EGC. The mean recovery was 29 ± 3% (SD), and the range was from 27 to 33%.
Study 1. After calibration of the probe, two 15-min fractions of
the outgoing dialysate were collected in all probes. Thereafter, one probe was
infused with two graded epinephrine concentrations (1 and 10 µM in Ringer
solution) for 30 min each, and a second probe was infused with two graded
isoproterenol concentrations (0.1 and 1 µM in Ringer solution) for 30 min
each. The concentrations of isoproterenol used were 10-fold lower than those
of epinephrine because this agonist is 10-fold more potent than epinephrine
toward -AR. A third probe was infused with Ringer solution throughout
the whole experimental period. After the end of the above-mentioned
perfusions, a hyperinsulinemic-euglycemic clamp was started and performed for
6 h. During hours 3 and 6 of the clamp, the perfusions using
epinephrine (probe 1) and isoproterenol (probe 2) were
performed with identical concentrations to those before the clamp. During the
time between the above-mentioned perfusions, the probes were infused with
Ringer solution. Dialysate samples were collected for each 10-min period
during the specific agent perfusions and for each 15 min during the rest of
the time.
Study 2. The experimental protocol was identical to that of study 1, except that, in probe 2, the graded concentrations of epinephrine (1 and 10 µM, i.e., the same concentrations as in probe 1) were infused each for 30 min with 100 µM phentolamine.
Control study (8 subjects). One probe was inserted into SCAAT, and the repeated perfusions, which used the same concentrations of epinephrine and the same time schedule as those described in the experimental protocol, were performed, except that the hyperinsulinemic-euglycemic clamp was not realized (Fig. 1). Control assays were also performed with phentolamine in combination with epinephrine without insulin administration in six subjects.
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Euglycemic-Hyperinsulinemic Clamp
The hyperinsulinemic-euglycemic clamp was performed by DeFronzo's method (9). A catheter for insulin and glucose infusion was inserted into an antecubital vein, and a second catheter for blood sampling was placed in a dorsal vein of the ipsilateral hand. The hand was kept in a warm box (60°C) to provide arterialization of venous blood. Priming plus continuous infusion of crystalline human insulin (1 mU·kg-1·min-1; Actrapid Human, Novo, Bagsvaerd, Denmark) was given for 6 h. Euglycemia (the fasting blood glucose concentration: 4.34 ± 0.07 mM) was maintained by a variable 15% glucose perfusion. The perfusion rate was determined by measurement of arterialized plasma glucose (Beckman Glucose Analyzer, Beckman Instruments, Fullerton, CA) every 5 min. Plasma concentrations of glucose, glycerol, and free insulin were analyzed in the basal state (mean value of 3 samples obtained 70, 65, and 60 min before the start of the clamp) and every 60 min during the clamp.
Drugs and Biochemical Determinations
The following drugs were used: isoproterenol hydrochloride (Isuprel, Winthrop), epinephrine hydrochloride (Epinephrine, Braun), and phentolamine methanesulfonate (Regitine, Ciba-Geigy, Rueil-Malmaison, France). Ethanol in dialysate and perfusate was determined with an enzymatic method (5); the intra-assay and interassay variabilities were 3.0 and 4.5%, respectively. Glycerol was determined in plasma and in dialysate by using an ultrasensitive radiometric method (6); the intra-assay and interassay variabilities were 5.0 and 9.2%, respectively. Plasma glucose was assayed with a glucose oxidase technique (Biotrol, Paris, France); the intra-assay and interassay variabilities were 1.5 and 5.1%, respectively. Nonesterified fatty acids were assayed with an enzymatic method (Unipath, Dardilly, France); the intra-assay and interassay variabilities were 1.1 and 1.6%, respectively. Plasma insulin was measured by using an Immunotech Insulin IRMA kit (Immunotech, Prague, Czech Republic); the intra-assay and interassay variabilities were 2.7 and 5.8%, respectively.
Data Analysis
All values are given as means ± SE. A statistical comparison of the values was performed by using two-way ANOVA for repeated measures with experimental conditions (control vs. euglycemic-hyperinsulinemic clamp) as factors of the analysis. Subsequently, the effects of drug perfusions were analyzed in each experimental condition by using two-way ANOVA with time as the factor of the analysis and followed by a Bonferroni-Dunnett post hoc test. The extracellular response curves were analyzed by using a paired t-test on the total integrated changes over baseline values [area under the curves (AUC)] by using the trapezoidal method. Values were considered statistically significant when P < 0.05. Statistical analyses were performed by using software packages (Statview 4.5 and SuperAnova 1.11, Abacus Concepts, Berkeley, CA).
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RESULTS |
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Lipolytic Response to Graded Epinephrine Perfusion in Subcutaneous Adipose Tissue: Control Study
After calibration of the probes, the two basal values of the EGC were similar. Addition of epinephrine to the perfusate caused a rapid dose-dependent increase in the EGC (Fig. 1). As soon as epinephrine was removed from the perfusate, EGC progressively decreased and returned to preperfusion values within 90 min. In the control investigation, i.e., without euglycemic-hyperinsulinemic clamp, the three successive epinephrine perfusion procedures at the two concentrations led to increases in EGC that were not different (Fig. 1A). The calculated AUC values were not significantly different among the three successive perfusions regardless of the epinephrine concentration used (Fig. 1B). Blood samples were taken before the start of each local perfusion for plasma glycerol concentration determination. No significant differences were found among the three values (48.4 ± 11.4, 48.0 ± 15.1, and 65.7 ± 11.4 µM, respectively). In six subjects, addition of phentolamine into the perfusate did not significantly modify the maximal increase of EGC in response to epinephrine perfusion during all three perfusions mentioned above (not shown).
Lipolytic Response to Graded Epinephrine or Isoproterenol Perfusion in Subcutaneous Adipose Tissue During Hyperinsulinemic-Euglycemic Clamp: Study 1
Immediately after the end of the first two graded epinephrine or isoproterenol perfusions in the probes, the hyperinsulinemic-euglycemic clamp was started. Before the euglycemic-hyperinsulinemic clamp, perfusion with addition of 1 and 10 µM epinephrine promoted an EGC increase (Fig. 2). Identical epinephrine perfusion during hour 3 of the clamp induced an EGC increase that was markedly lower (P = 0.05) than that before the clamp (Fig. 2A). Finally, the reduction of the epinephrine-induced increase in EGC was even more pronounced during hour 6 of the clamp (P < 0.05; Fig. 2B). From the calculated AUC values, it appeared that the effect of 1 and 10 µM epinephrine perfusion was reduced by 72 and 63%, respectively, during hour 3 of the clamp and by 81 and 70% during hour 6 of the clamp.
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In the same protocol, graded concentrations of isoproterenol (0.1 and 1 µM) were infused in the second probe at the same time intervals (Fig. 2). Before the clamp, isoproterenol induced an increase in the EGC values. The maximal EGC values were close to those observed with the 10-fold higher concentration of epinephrine. During hour 3 of the clamp, isoproterenol-induced EGC increase was lower than before the clamp (P < 0.05). Finally, the reduction of the isoproterenol-induced increase in EGC was more pronounced during hour 6 of the clamp (P < 0.05). When the calculated AUC values were used (Fig. 2B), the effect of the 0.1 and 1 µM isoproterenol perfusion was reduced by 41 and 28%, respectively, during hour 3 of the clamp and by 48 and 44%, respectively, during hour 6 of the clamp. The reductions of the isoproterenol-induced increases in EGC were lower when compared with the reductions for epinephrine-induced increases at the same time interval: the effect of 0.1 µM isoproterenol was compared with that of 1 µM epinephrine (P < 0.05 at hour 3 and P < 0.01 at hour 6) and that of 1 µM isoproterenol with 10 µM epinephrine (P < 0.01 at hour 3 and P < 0.04 at hour 6).
Comparison of the Lipolytic Response to Graded Perfusion of Epinephrine Alone or Associated with Phentolamine During Hyperinsulinemic-Euglycemic Clamp: Study 2
Two probes were used; both were supplemented with epinephrine solutions that were identical in concentrations and time schedule of administration to those of study, but in probe 2, 100 µM phentolamine was added to all the epinephrine solutions. Before the euglycemichyperinsulinemic clamp, addition of phentolamine into the perfusate did not significantly modify the maximal increase of EGC in response to epinephrine perfusion (Fig. 3). During hour 3 of the euglycemic-hyperinsulinemic clamp, the epinephrine perfusion resulted in a markedly decreased response when compared with that before the clamp. During the same time, the reduction of the lipolytic response was lower in the phentolamine-containing probe. The same situation also occurred during hour 6 of the clamp. When the responses were evaluated by using AUC (Fig. 3B), it appeared that, during hour 3 of the clamp, the epinephrine effect was reduced by 67 and 59% in the probe containing 1 and 10 µM epinephrine, respectively. It was less reduced by 35 and 47% in the probe containing the phentolamine in addition to epinephrine. The difference between the responses in the two probes was significant. The same difference was shown during hour 6 of the clamp, where the effect of the sole epinephrine perfusion was reduced by 75 and 67% and the effect in the phentolamine-containing probe was reduced by only 19 and 40%, respectively. The difference between the responses in the two probes remained significant. Thus, during the euglycemic-hyperinsulinemic clamp, the responses of EGCs to epinephrine perfusion were significantly less reduced in the presence of phentolamine than in control probes containing epinephrine alone.
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Extracellular and Plasma Glycerol Concentrations During the Euglycemic-Hyperinsulinemic Clamp
The changes of EGC during the euglycemic-hyperinsulinemic clamp were evaluated in a probe that was infused with Ringer solution throughout the experimental protocol (Fig. 2). Dialysate samples were taken at the same time intervals as in the other probes. Blood samples were taken 5 min before the clamp and at minutes 180 and 360 of the clamp for plasma glycerol concentration determination. We observed a progressive decrease of EGC (246 ± 16.3, 194 ± 36, and 150 ± 37 µM) and plasma glycerol concentrations (34.8 ± 3.9, 9.7 ± 2.5, and 9.2 ± 2.1 µM) during the euglycemichyperinsulinemic clamp
Modifications of Regional ATBF
The changes in the local adipose tissue microcirculation were evaluated by using the method based on the measurement of ethanol washout. Ethanol ratio was calculated as reported in METHODS and taken as an index of ethanol washout. The higher ethanol ratio corresponds to the lower ethanol washout, and this reflects the lower regional ATBF. The average ethanol ratios measured during the last 10-min period of each graded perfusion of catecholamines are presented in Fig. 4. In the control study, there were no differences in the epinephrine-induced increase (at 10 µM) in the ethanol ratio during the course of the protocol despite the fact that the increase did not reach a level of significance during the third perfusion (Fig. 4). In the study with clamp (probe 2), the epinephrine-induced increase in the ethanol ratio (10 µM) before the clamp was not different from increases during the clamp. Both isoproterenol perfusion (probe 1) and the perfusion of epinephrine combined with phentolamine (probe 2) did not induce a significant decrease in ethanol ratios during the clamp.
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DISCUSSION |
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Lipolysis in human fat cells is mainly controlled by insulin and
catecholamine. Catecholamines (i.e., epinephrine and norepinephrine) promote
the activation of both lipolytic -ARs and antilipolytic
2-ARs. The occurrence of an antilipolytic potency of
catecholamines, particularly epinephrine, which exhibits a higher affinity for
the
2-AR, is noticeable in human subcutaneous adipocytes.
Consequently, epinephrine is less efficient in stimulating lipolysis than a
selective catecholamine that possesses only the
-AR potency. Moreover,
the antilipolytic action of epinephrine is particularly expressed in
subcutaneous adipocytes from obese subjects, and we have previously
demonstrated that, in obese subjects, the activation of the
2-AR strongly reduces the lipolytic effect of physiological
catecholamines during exercise
(30). To assess the hypothesis
of a balanced activity of
- and
2-ARs (when the
involvement of the
-AR pathway is lowered in adipose tissue, the
antilipolytic
2-AR component is strengthened), we compared
the effect of insulin administration on the lipolytic effect of epinephrine
with isoproterenol explored in situ.
At the beginning of the present studies, we verified the absence of desensitization when catecholamine perfusions were performed. The interval between the various epinephrine perfusions has been selected to prevent (or limit) the incidence of desensitization and limit its interference with the effect of insulin. Repeated intravenous perfusions of epinephrine (28) or microdialysis perfusion (24) could lead to desensitization of catecholamine-induced lipolysis in human adipose tissue. This phenomenon was not found in rat adipose tissue when norepinephrine was infused into microdialysis probes at 1-h intervals (8). In fact, it occurs in human adipose tissue when catecholamine perfusions are applied at intervals of 1 h (24) or less (28). We verified the absence of desensitization when catecholamine perfusions were realized in an interval of 2 h. In the control study without euglycemic-hyperinsulinemic clamp, the repeated perfusions of epinephrine into the probes were realized, and they resulted in the same EGC increase for both epinephrine concentrations (Fig. 1).
It has previously been shown that the in vitro incubation of fat cells with
insulin significantly reduces the lipolytic effect of isoproterenol
(10). This effect is
classically attributed to the insulin-dependent activation of
phosphodiesterase, an intracellular enzyme that hydrolyzes cAMP. An additional
explanation involves cross-regulation between insulin and
2-AR, i.e., growth factor tyrosine kinase receptors, like
insulin receptor, directly phosphorylating a G protein linked to the
2-AR (3). This
mechanism probably leads to internalization of
-AR into the cytoplasmic
compartment since binding studies have revealed a decrease in
-AR in the
plasma membrane of human fat cells treated with insulin
(10). It has also been shown
that insulin has no effect on the binding of
2-AR-selective
radioligands in human fat cells
(10) and on the antilipolytic
effect of the selective
2-AR adrenergic agonist UK-14304
(personal data). So, in insulin-treated fat cells, the
-/
2-AR balance is modified in favor of the
2-AR antilipolytic pathway in human fat cells.
At the concentration used, insulin inhibits spontaneous lipolysis in the
SCAAT and lipid mobilization in the fasted subjects. Moreover,
hyperinsulinemia reduced the isoproterenol-induced lipolysis
(Fig. 2). Isoproterenol
exhibits an 10-fold higher affinity for
-ARs than epinephrine. Thus
the chosen concentrations of epinephrine infused in the probes were 10-fold
higher than those of isoproterenol to obtain a similar increase in EGC with
both amines. In such conditions, before the euglycemic-hyperinsulinemic clamp,
the increase in EGC was similar with the two catecholamines. The reduction of
epinephrine-induced lipolysis during the clamp was significantly higher than
that of isoproterenol-stimulated lipolysis for the corresponding
concentrations. The blunting effect of insulin on the epinephrine-induced
changes in subcutaneous EGC (an index of in situ lipolysis) could result from
multiple mechanisms. The first one could be due to phosphodiesterase
activation and/or phosphorylation of the G protein linked to the
-AR or
the
-AR itself by insulin, and the second one could result from the
inhibition of the formation of cAMP through activation of fat cell
2-ARs (7,
19). The latter mechanism
could be involved in differences observed between the effects of
hyperinsulinemia on the lipolytic effects of epinephrine alone and epinephrine
coupled with phentolamine. When phentolamine (an
1-/
2-AR antagonist) was infused in the
probe together with epinephrine, the EGC response was higher during the clamp
than with epinephrine alone, and, consequently, the reduction in lipolytic
effect promoted by epinephrine, during the euglycemichyperinsulinemic clamp,
was clearly less pronounced. It is noticeable that before the
euglycemic-hyperinsulinemic clamp, phentolamine alone did not significantly
change the epinephrine-induced increase in EGC. The latter observation
suggests that, in the presence of low plasma insulin concentrations, the
2-AR antilipolytic effect is not apparent. This is in
disagreement with a previous report showing that phentolamine potentiates both
spontaneous and norepinephrine-stimulated lipolysis
(2) in a similar microdialysis
protocol. However, in several other studies, the enhancing effect of
phentolamine on spontaneous glycerol release has not been found
(15,
21). In fact, it was shown
that the potentiating effect of phentolamine resulting from
2-AR blockade in SCAAT could be discerned clearly during
exercise when the activity of the adrenergic system is increased
(21).
During interpretation of lipolysis data based on glycerol concentrations in
dialysate outflowing from the microdialysis probe, the regional ATBF must be
taken into account. It has been demonstrated that the EGC in human adipose
tissue is influenced by the local blood flow
(4,
11,
13). In the present study, it
was not possible to evaluate ATBF changes induced by the perfusion of
pharmacological agents through the probe by the conventional technique, i.e.,
the 133Xe washout; the space of diffusion of the perfused agents,
and consequently the volume in which the ATBF changes are produced in SCAAT
are very narrow and different from the distribution volume of xenon injected
directly into SCAAT. Consequently, the 133Xe-washout method
reflects the blood flow in larger and not identical volumes of adipose tissue.
Thus a method based on the evaluation of ethanol washout, measured by using
ethanol ratio, was used for assessment of changes in adipose tissue
microcirculation. The increase in ethanol ratio reflects a decrease in ATBF
(12). The ethanol method does
not enable the corrections of individual EGC for ATBF values, but, reflecting
the variations in ATBF, it provides a possibility to assess whether and in
which sense the changes in EGC were influenced by the changes in ATBF. In this
study, epinephrine induced a decrease in ATBF that was significant at the
concentration of 10 µM (Fig.
4). This finding could suggest that the epinephrine-induced
increase in EGC is enhanced because of the reduction of local blood flow and,
consequently, that the increase in the rate of lipolysis is over estimated.
During the euglycemic-hyperinsulinemic clamp, the epinephrine effect on ATBF
was not modified. This finding suggests that the reduction in
epinephrine-induced increase of EGC, observed during
euglycemic-hyperinsulinemic clamp, was not influenced by the changes in ATBF
and thus reflects the true inhibition of lipolytic processes in adipose
tissue. Insulin by itself, at the concentrations used, does not change ATBF as
previously shown (29). When
epinephrine was perfused with phentolamine, a tendency toward a nonsignificant
increase in ethanol washout (i.e., an increase in local ATBF) was observed.
Thus the potentiating effect of phentolamine on an epinephrine-induced
increase in EGC during euglycemic-hyperinsulinemic clamp could be attributed
to the blockade of fat cell 2-ARs, and the slight
phentolamine-induced vasodilatation could lead only to underestimation of
potentiating action of phentolamine on epinephrine-induced lipolysis.
In summary, the present study demonstrates that hyperinsulinemia reduces
the lipolytic effect of catecholamines in adipose tissue. The results suggest
that several mechanisms are involved in the resulting insulin-induced blunting
of the epinephrine-induced lipolysis in situ. First, the -AR-dependent
lipolytic pathway is reduced, presumably through the activation of
phosphodiesterase-3B and by the desensitization of the
-AR-dependent
mechanisms from insulin. These mechanisms could explain the reduction of the
effect of isoproterenol. Second, the activation of the antilipolytic
2-AR-dependent pathway by epinephrine could contribute to
counteraction of the
-AR-dependent lipolytic pathway, which would
explain the alterations that occur in the action of epinephrine. The present
study suggests that hyperinsulinemia contributes to alterations of adipose
tissue characteristics that were described in insulin resistance (such as
upper body obesity) and suggests the mechanisms by which
- and
2-AR-mediated pathways are involved in these alterations. It
is not the only pathway related to hyperinsulinemia-induced actions, and its
physiological importance, compared with other pathways, remains to be firmly
established. Further studies investigating in vivo the relative contribution
of
- and
2-AR-mediated pathways in adipose tissue
metabolism in conditions related to altered levels of plasma insulin
(physiological: nutrition; pathological: obesity, diabetes) are warranted.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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