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Experimental Studies |
Laboratory of Experimental Medicine and Department of Endocrinology, Hôpital Erasme, University of Brussels, Brussels, Belgium
Address all correspondence and requests for reprints to: Dr. Françoise Féry, Laboratory of Experimental Medicine, University of Brussels, 808 route de Lennik, B-1070 Brussels, Belgium.
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Abstract |
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) from
baseline were recorded in Acipimox vs. placebo studies:
FFA, -0.26 ± 0.08 vs. +0.29 ± 0.06 mmol/L
(P < 0.001); glucose, -12 ± 2
vs. -12 ± 1 mg/dL (P > 0.05);
glucose production, +16 ± 5 vs. -15 ± 3
mg/min (P < 0.001); C peptide, -1.11 ±
0.10 vs. -0.66 ± 0.10 ng/mL (P <
0.001); glucagon, +64 ± 25 vs. +21 ± 9 pg/mL
(P < 0.05); GH, +37 ± 9 vs.
+4 ± 2 ng/mL (P < 0.007); cortisol,
+37 ± 25 vs. -30 ± 26 ng/mL
(P < 0.04). Acipimox inhibited fat oxidation
(-18 ± 4 vs. +19 ± 4 mg/min;
P < 0.001) and enhanced carbohydrate oxidation
(+18 ± 8 vs. - 24 ± 11 mg/min;
P < 0.02). Protein catabolism calculated over the
8-h study period was significantly stimulated (+5.7 ± 2.5
vs. -1.9 ± 1.7 g/8 h; P < 0.02).
During the Acipimox studies, the increased protein breakdown could
theoretically account for about 75% of the increased glucose
production. Thus, contrary to current opinion, FFA suppression
stimulates glucose production and whole body glucose disposal in normal
overnight fasted subjects. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Another point of controversy regarding the FFA-glucose interrelationships relates to the role played by circulating FFA in the regulation of gluconeogenesis and glucose production. With a few exceptions (13), most in vitro studies have shown that FFA stimulate hepatic gluconeogenesis from various precursors in the rat (14, 15, 16). However, important species differences exist, and FFA have no effect on gluconeogenesis in isolated hepatocytes from the newborn pig (17) and inhibit gluconeogenesis and glucose production in perfused liver from the dog (18), cat (19), and guinea pig (20). In human experiments, it has been observed that gluconeogenesis from lactate is stimulated by increased FFA levels (21) and inhibited by an acute antilipolytic treatment (22), but in these latter two studies, as in others (2, 3, 9, 10), the changes in FFA levels had no effect on basal hepatic glucose output. Recent data from our laboratory tend to contradict these observations. We have reported (23) that FFA suppression induces an increase, rather than a decrease, in gluconeogenesis and enhances liver glucose production in 4-day fasted subjects, but it is not known whether this applies to the overnight fasted state.
It is thus fair to admit that the role played by FFA availability in the regulation of whole body glucose metabolism in overnight-fasted subjects remains unclear. This question is examined in the present study, which analyzes the effect of a pharmacological suppression of FFA levels on glucose production, uptake, and oxidation measured with the combined use of radiolabeled glucose and indirect calorimetry. The effect of antilipolysis was studied over an extended period of 8 h, because there is evidence that FFA-glucose interactions in vivo proceed at a relatively slow rate (5, 23).
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Materials and Methods |
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For each test, the subject reported at the metabolic unit at 0700 h after an overnight fast of 12 h. A Teflon catheter was inserted into an antecubital vein for infusion, and another catheter was inserted in a dorsal vein of the hand opposite the site of infusion for sampling of arterialized blood. For this purpose, the hand was maintained in a heated electrical pad during the entire study. After insertion of the catheters, a primed constant infusion of [3-3H]glucose diluted in saline and sterilized through a 0.22 µm Millipore filter (Millipore Corp., Bedford, MA) was administered for 10.5 h. The priming dose represented 125 times the amount infused per min. Total radioactivity administered amounted to approximately 300 µCi.
Each subject participated in two tests performed in a random order at a 2- to 3-week interval. In one test, after a basal period of 2.5 h, the subjects received Acipimox administered as one capsule of 250 mg at 0 and 2 h and two capsules at 4 and 6 h. In the other test, Acipimox was replaced by placebo.
Blood samples were obtained every 15 min during the last hour of the basal period, every hour for the following 6 h, and every 30 min thereafter. The subjects collected their urine during the night before the test and voided before the start of the study, and urine was collected again at the end of the experiment. Respiratory gas exchanges for indirect calorimetry were monitored during the basal period and thereafter for a 30-min period every hour using a Deltratrac Metabolic Monitor (Datex, Helsinki, Finland).
Analytical methods
Blood samples were collected in heparinized syringes and transferred to tubes kept on ice. Those intended for measuring unlabeled and labeled glucose and lactate contained NaF, and those used for measuring glucagon concentration contained aprotinin. After centrifugation at 4 C, plasma was stored at -20 C until assayed. The plasma concentrations of FFA, glucose, lactate, glycerol, urea, insulin, C peptide, glucagon, GH, and cortisol and urinary total nitrogen content were determined as recently described (23). Plasma [3H]glucose concentrations were determined by scintillation spectrometry on evaporated Somogyi filtrates reconstituted with water. All determinations were performed at least in duplicate.
Calculations
The rates of appearance (Ra) and disappearance (Rd) of glucose were determined by the nonsteady state equations of Steele (24) using a glucose distribution volume of 200 mL/kg and a pool fraction of 0.65. The MCR of glucose was calculated as the ratio between Rd and the plasma glucose concentration. Carbohydrate and lipid oxidation rates were determined from CO2 production, O2 consumption, and protein oxidation (25). The latter was estimated from urinary nitrogen excretion and changes in the urea nitrogen pool size (26). To avoid underestimation of carbohydrate oxidation related to gluconeogenesis from amino acids, it was assumed that the totality of the degraded proteins was converted to glucose with subsequent oxidation of the newly formed glucose (27). A factor of 0.57 for the conversion of proteins to glucose was used in this calculation. Finally, carbohydrate oxidation was further corrected by adding the equivalent of 10% by mass of net lipid oxidation to account for gluconeogenesis from glycerol (27).
Statistical analysis
Results obtained at the various time points of the studies were
expressed as changes (±SE) from the mean basal values
(Figs. 1
and 2). Statistical analysis was
performed using the Computer Program Superanova (Abacus Concepts,
Berkeley, CA). Data were analyzed by a two-factor ANOVA (treatment and
time) for repeated measures of both factors. Whenever the F ratio was
less than 0.05, a pairwise comparison between the two treatments was
performed at the various times using modified Student’s t
test. The average values recorded during the last 2 h of the
studies were compared by a paired t test (Table 2).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the basal values of all
parameters tested were similar for the two studies, thus allowing
comparison between the effects of Acipimox and placebo on the basis of
changes from mean baseline values. As shown in Figs. 1
and 2, FFA
levels were dramatically reduced after Acipimox. The average fall
observed from the second to the sixth hour of treatment exceeded 90%
of initial value, but levels rose slightly during the last 2 h.
This tendency to escape the action of the drug was also observed for
glycerol concentrations. Fat oxidation followed the same pattern; a
maximal inhibition of about 40% was observed 5–6 h after starting the
drug treatment. FFA and glycerol levels and fat oxidation rate rose
significantly during the placebo studies.
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13%) during the control and the active studies (Fig. 1
and Table 2). However, the changes in glucose kinetics
(Fig. 2 and Table 2) were markedly different; glucose Ra and Rd rose by
about 10% during the 8 h of the Acipimox experiments, but
decreased by about 10% during the placebo studies; the difference
between the two curves was highly significant (P <
0.001). The MCR of glucose was stimulated by about 20% during the
antilipolytic treatment and remained essentially unchanged in the
control experiments (P < 0.001). The changes in
glucose oxidation were the mirror image of those of fat oxidation.
Thus, the increase in glucose oxidation induced by Acipimox attained
its highest value (+30%) after about 5 h of treatment, but
tended to be less marked thereafter. There was a highly significant
difference between the glucose oxidation curves observed during the
two experimental conditions (P = 0.005).
Antilipolysis was associated with several hormonal changes (Fig. 1 and
Table 2). Insulin levels fell gradually under both experimental
conditions. Suppression was slightly more marked during the last 4
h of the Acipimox vs. placebo treatments, but the difference
was not statistically significant. The difference was, however, highly
significant with regard to C peptide levels (P =
0.007). The glucagon concentrations were significantly increased
(P < 0.01), and the insulin/glucagon ratios were
significantly decreased (P = 0.015) during active
vs. control experiments. Finally, antilipolysis induced a
significant rise in GH (P = 0.005) and in cortisol
levels during the last 2–3 h of active treatment, but the statistical
significance was borderline (P = 0.049) for the latter
hormone.
The changes in nitrogen metabolism are displayed in Table 3. Compared to placebo, Acipimox increased urinary
nitrogen excretion, but not to a significant extent. The urea nitrogen
pool size (P < 0.05) and total urea nitrogen
production (P < 0.02) were significantly augmented.
Thus, on the average, total protein breakdown increased by 5.7 ±
2.5 g during the 8 h following Acipimox treatment and
decreased by 1.9 ± 1.7 g after placebo; the difference
between the two treatment regimens was statistically significant
(P < 0.02).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the active study, the administration of Acipimox markedly lowered the rates of lipolysis and whole body fat oxidation during the entire experimental period, but some escape from the action of the drug was noticed during the last 2 h despite an increase in the dosage of the drug. In agreement with other studies (8, 11, 28, 29), we observed that antilipolysis reduces insulin and C peptide levels and increases the concentrations of glucagon, cortisol, and GH. As these hormonal changes occur essentially at the end of the test, their lipolytic effect is probably responsible for the escape phenomenon.
The first consequence of FFA suppression is a stimulation of whole body glucose uptake. As glucose levels are not affected by the drug, the increased uptake is entirely related to stimulation of the MCR, which reflects an increased capacity of tissues to extract circulating glucose. As the increase in glucose oxidation exceeds (although not significantly) that in uptake, our data suggest that the augmented glucose disposal is entirely accounted for by increased oxidation. However, such an assertion should be made very cautiously, because glucose oxidation was measured by indirect calorimetry, and therefore, it includes oxidation of muscle glycogen, which could have been stimulated by the reduction in FFA availability (30). If this were to occur, the measured increase in carbohydrate oxidation would overestimate that in circulating glucose.
The observed increase in the rates of whole body glucose uptake, metabolic clearance, and oxidation cannot be explained by the hormonal changes. The increased uptake is probably mainly due to the operation of the glucose-fatty acid cycle at the level of skeletal and heart muscles, as documented by Nuutila et al. (12). If it is assumed that skeletal muscle glucose uptake represents about 20% of whole body glucose uptake under postabsorptive conditions (31) and that quantitatively tissues other than muscle play a negligible role in the process, it can be calculated that muscle glucose disposal must have been approximately doubled to account for the 25% increase in overall glucose consumption.
The reason why, in contrast with other studies (6, 9), we were able to
demonstrate an increase in whole body glucose uptake after
antilipolytic treatment in the postabsorptive state probably lies in
the fact that lipolysis was more markedly reduced. Indeed, the average
FFA concentration fell to as low as 0.03 mmol/L after 2 h of
treatment and remained below 0.2 mmol/L until the end of the study.
These levels are much lower than those obtained in comparable studies
(6, 9, 12), possibly because our volunteers had fasted for only 12
h before the test and had low initial levels of FFA (0.4 mmol/L). It
has been shown (12) that the negative relationship between FFA levels
and muscle glucose uptake is curvilinear, so that the stimulatory
effect of a given fall in FFA levels on muscle glucose uptake is much
larger in the low FFA range and should, therefore, more markedly
influence whole body glucose uptake.
A second aspect of our data is that FFA lowering is associated with a stimulation of glucose Ra. It is possible that this phenomenon represents a direct consequence of FFA suppression on hepatic glucose output as observed in several other animal species (18, 19, 20). However, this is unlikely, because in humans, gluconeogenesis seems to be inhibited by an antilipolytic treatment, at least in type 2 diabetic patients (22). Therefore, the rise in Ra is probably an indirect consequence of antilipolysis mediated by the various hormonal changes, which are all able to stimulate hepatic glucose production.
The source of the extra glucose formed cannot be precisely determined from our experiments. It could involve a stimulation of hepatic glycogenolysis, as documented in mice after acute nicotinic acid treatment (30), and/or a stimulation of gluconeogenesis. As far as this latter possibility is concerned, our data indicate that antilipolysis induces an increase in protein breakdown that could account for about 75% of the rise in glucose Ra if it is assumed (27) that the carbon skeleton of the catabolized proteins was totally converted to glucose. Whether the increase in protein catabolism is mediated by the hormonal changes or by a direct effect of FFA shortage on muscle protein metabolism (32) remains unsettled.
The overall metabolic and hormonal adaptations occurring in response to acute antilipolysis can thus be viewed as follows. The reduction in FFA availability induced by Acipimox inhibits whole body and presumably muscle fat oxidation. By virtue of the operation of the glucose-fatty acid cycle, muscle carbohydrate oxidation is stimulated, inducing an increase in the extraction of circulating glucose. On the other hand, the lowering in FFA levels induces several hormonal changes that could be responsible for the increases in both protein breakdown and overall glucose production. This increase in production exactly compensates the increased uptake, so that the changes in glycemia are not different from those occurring in the placebo studies. The global effect of this regulation is to maintain adequate fuel supply to muscle and possibly other tissues despite the reduced FFA availability. Under our experimental conditions, there is no reason to suspect the occurrence of any change in brain metabolism that does not rely on FFA.
It is interesting to compare the present results with those of a recent
study (23) in which we applied the same protocol to 4-day fasted
subjects. Under these conditions, antilipolysis decreased not only the
levels of FFA, but also those of ketone bodies, which were initially
markedly elevated by the fast. The concentrations of the
glucoregulatory hormones, the rate of protein breakdown, and the rates
of glucose production and uptake were modified by the antilipolytic
treatment in the same direction as that observed in the present study
(Table 4). However, in contrast to the present results,
the MCR of glucose remained unchanged, and the rise in the Rd was
entirely mediated by the increase in the glucose concentration. Thus,
it seems that the glycemic response to antilipolysis in normal subjects
is critically dependent on the composition of brain energy supply;
glucose levels rise only if antilipolysis induces a shortage of brain
fuel supply as is the case in the hyperketonemia due to fasting, but
not in the overnight fasted state. This is an argument in favor of an
important role of the central nervous system in determining the
glycemic set-point.
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Acknowledgments |
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Footnotes |
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Received September 26, 1996.
Revised November 4, 1996.
Accepted November 19, 1996.
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References |
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