Mechanisms of glucose intolerance during triglyceride infusion

V. Rigalleau1, M. Beylot2, C. Pachiaudi3, C. Guillot4, G. Deleris4, and H. Gin1

1 Service de Nutrition et Diabétologie, Hôpital Haut-Lévêque, 33600 Pessac; 2 Institut National de la Santé et de la Recherche Médicale U-499, Faculté de Médecine Laennec and 3 Centre de Recherche en Nutrition Humaine de Lyon, 69372 Lyon; and 4 Laboratoire de Chimie Bioorganique, Université Victor Segalen, 3300 Bordeaux, France

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Lipid infusions may affect glucose tolerance by effects on glucose production or utilization. We performed double-labeled oral glucose tolerance tests with and without a lipid infusion in eight normal subjects. During the lipid infusion, plasma glucose and insulin levels were higher, showing some insulin resistance. The increased glucose level was due to a higher total glucose appearance rate, partly reproducible by a control infusion of glycerol [saline 1,181 ± 71 mg · kg-1 · 330 min-1 vs. lipid 1,388 ± 100 (P < 0.05) vs. glycerol 1,276 ± 126 (NS)]. The tracer-determined appearance rate of exogenous glucose was higher with lipid infusion but was probably overestimated because of higher 13C recycling into glucose. Residual systemic glucose production was increased but was reproducible by the glycerol infusion. Total glucose disposal was increased. This was observed despite a lower stimulation of total glucose oxidation as measured by indirect calorimetry, whereas oxidation of exogenous glucose was normal after correction for the lipid-induced modification of excretion rate of 13CO2. Accordingly, glucose nonoxidative disposal was increased. These moderate modifications of glucose metabolism (increased appearance, increased nonoxidative disposal, and lower total oxidation) have been reported in starvation-induced or spontaneously impaired glucose tolerance. Further impairment, especially decreased nonoxidative glucose disposal, seems to be required to produce non-insulin-dependent diabetes mellitus.

oral glucose tolerance; Randle cycle; systemic glucose production; glucose disposal; glucose recycling

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

MORE THAN THIRTY YEARS AGO, Randle et al. (33) showed that increased availability of lipid substrates reduced glucose utilization, which was thought to play a role in insulin-resistant states, such as obesity, diabetes, or starvation. Numerous studies examining the influence of lipid infusions on glucose metabolism have since been devoted to the "glucose-free fatty acid cycle" in humans.

Lipid infusions have been found to have little effect in the postabsorptive state (8) unless insulin secretion is disrupted as in normal subjects by infusion of somatostatin (5) or in patients with non-insulin-dependent diabetes mellitus (NIDDM) (35). On the other hand, both insulin-stimulated glucose oxidation and nonoxidative disposal (6, 7) are impaired by infusion of lipids during euglycemic hyperinsulinemic clamp. However, these observations of lipid-induced insulin resistance in situations of euglycemia do not necessarily imply that lipid-carbohydrate interactions play a significant role in the alterations of glucose metabolism observed in the postprandial state of insulin-resistant subjects, since plasma glucose levels are increased in the latter case. Effects of lipid infusions may be more relevant if tested under the conditions of the oral glucose tolerance test (OGTT), which is closer to the postprandial state.

The influence of a lipid infusion on glucose tolerance has been studied by Rousselle et al. (37) and Kruszynska et al. (23) in normal subjects and Meylan et al. (26) in obese subjects. In these studies, lipids inhibited glucose oxidation, although glucose nonoxidative disposal was increased, presumably due to the slight increase in glycemia. However, indirect calorimetry may lead to erroneous conclusions concerning glucose oxidation when lipids are infused, since they stimulate gluconeogenesis (9, 10), which could result in an underestimate of glucose oxidation if amino acids are the main neoglucogenic precursors used (14). The study from Kruszynska et al. (23) suggested that lipid infusion also induced a defective suppression of systemic glucose production (SGP), as observed during clamp studies with lipid infusion (24), but the potential role of the glycerol infused was not evaluated in this observation.

In the present study, we performed double-labeled OGTTs with a naturally 13C-enriched oral load (cornstarch glucose; Ref. 41) and a dideuterated glucose infusion in eight normal volunteers. Each subject was studied twice, once during a saline infusion (controls) and once during a lipid infusion. To determine the influence of glycerol in the infused lipid emulsion on glucose appearance, four subjects were studied a third time, during a glycerol infusion. Four subjects also underwent a lipid infusion without OGTT to assess the contribution of 13C from infused lipids to expired 13CO2. Using this methodology, we could determine the respective contribution of modifications in SGP, glucose oxidation, and nonoxidative disposal to lipid-induced alteration of glucose tolerance.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. D-[6,6-2H2]glucose (99 atom% excess) was obtained from the Commissariat à l'Energie Atomique (Gif-sur-Yvette, France). It was checked to be sterile and pyrogen free and was dissolved in sterile normal saline solution before administration. Pure cornstarch glucose was obtained from Aguettant (Lyon, France). The triglyceride emulsion (Ivélip, 20%) was obtained from Cernep Synthélabo (Montargis, France). It contained 200 g/l long-chain triglycerides (fatty acids: 11% palmitic acid, 5% stearic acid, 26% oleic acid, 50% linoleic acid, 7% linolenic acid, 0.5% gadoleic acid, and 0.5% erucic acid) and 25 g/l glycerol to stabilize the emulsion. The 100 g/l glycerol solution was obtained from the Pharmacie Centrale des Hôpitaux de Bordeaux. Gas exchanges were measured using a Deltatrac metabolic monitor (Datex, France).

Subjects. Eight subjects were studied twice by doubly labeled OGTTs, once under infusion of saline (control tests) and once under a lipid infusion (Ivélip test) 1 mo later. Four of these subjects had two more tests: one OGTT under a glycerol infusion and one lipid infusion (Ivélip) without OGTT. They were normal healthy volunteers (5 men and 3 women, age 23 ± 2 yr, body weight 63.2 ± 2.6 kg, body mass index 21.5 ± 0.4). None of them had a family history of diabetes or were receiving medication. They were requested to consume their normal diet but to avoid food containing cane sugar, cornstarch, or exotic fruits for 1 wk before and between the tests. They gave their written consent to the study after being informed of its nature, purpose, and potential risks. The protocol was approved by the Ethical Committee of Edouard Herriot Hospital (Lyon, France).

Protocol. Figure 1 shows the study design. All subjects were studied in the postabsorptive state after a 12-h overnight fast in the metabolic unit of the Service de Nutrition-Diabétologie (Hôpital Haut-Lévêque, Pessac, France). A retrograde catheter was inserted in a dorsal hand vein and kept in a hot blanket (55°C) to collect arterialized venous blood. A forearm vein of the controlateral arm was catheterized to infuse D-[6,6-2H2]glucose and saline, Ivélip, or glycerol.


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Fig. 1.   Study design.

A priming dose of D-[6,6-2H2]glucose (6 mg/kg) was administered at 0800, and D-[6,6-2H2]glucose was then infused at a constant rate (0.06 mg · kg-1 · min-1) using an electric syringe (Harvard, Les Ulis, France) for 450 min. Half an hour later, a constant-rate infusion (0.015 ml · kg-1 · min-1 as used in previous studies; Refs. 24, 35) of saline (control tests) or Ivélip (Ivélip tests) began and was continued until the end of the test by using a volumetric infusion pump (Harvard). Because heparin has its own effects on insulin binding and biological activity, we did not use it in combination with the lipid infusion (21). Four subjects underwent a third test with a constant-rate infusion (0.004 ml · kg-1 · min-1) of a 100 g/l glycerol solution to test the effect on glucose appearance of the glycerol used in the triglyceride emulsion. The first 120 min of the tests (from times -120 to 0) were allowed for isotopic equilibration and measurement of postabsorptive glucose turnover rate. Thereafter, a dose of 1 g/kg of naturally 13C-enriched cornstarch glucose, diluted in water (30 g/100 ml), was ingested.

In four subjects, the effect of the Ivélip infusion alone (0.015 ml · kg-1 · min-1 during 420 min, without OGTT) on 13C enrichment of expired CO2 was determined to allow calculation of a corrected exogenous glucose oxidation rate (Gexo,corr).

Throughout the test, samples of blood and of expired gases were drawn for determination of metabolites and hormone levels and for isotopic enrichments. Except during the glycerol infusion (which was designed to study glucose appearance), gaseous exchange was measured continuously using the Deltatrac metabolic monitor, with a 5-min pause every 30 min to collect expired gases for isotopic enrichment. Urine samples were collected at times -120, 0, and 330 for urinary nitrogen excretion determination.

Analytical procedures. Plasma glucose, free fatty acids (FFA) (25), glycerol, and triglyceride concentrations were determined enzymatically (2). Urinary nitrogen was determined by the Kjeldahl method. Insulin and C-peptide levels were measured by RIA. Plasma D-[6,6-2H2]glucose enrichment was measured by conventional selected ion-monitoring gas chromatography-mass spectrometry (Hewlett-Packard 5971A-MSD, Paris, France) as described by Bier et al. (3). Plasma and ingested [13C]glucose isotopic enrichment was measured by gas chromatography-isotope ratio mass spectrometry (Sira 12, VG Instrument, Middlewich, UK) as described by Tissot et al. (41). 13C enrichment of expired CO2 was determined on the dual-inlet isotope ratio mass spectrometer described by Guilluy et al. (19). The 13C-to-12C ratios of the samples were expressed as differences from the International PDB standard according to the formula, partial 13Cp × 1,000 = [(13C/12C sample - 13C/12C standard)/(13C/12C standard)] × 1,000, and was transformed to atom percent excess (APE) for ingested and plasma glucose as previously described (41).

To determine the relative 13C abundance of glucose, glycerol, and lipid emulsions, samples of these solutions were combusted at 1,020°C. The CO2 was separated using a liquid N2 trap and analyzed for 13C abundance. The isotopic abundance measured after oxidation was -12.6, -30.2, -29.5, and -28.7 partial 13Cp × 1,000 for the glucose, glycerol from the Pharmacie Centrale des Hôpitaux de Bordeaux, glycerol used for stabilization of Ivélip (furnished by Cernep Synthélabo), and total Ivélip, respectively.

Calculations. Rates of total glucose appearance (RaT) and disappearance (RdT) were calculated from the enrichment of blood glucose by D-[6,6-2H2]glucose, using the steady-state equation in the postabsorptive state and the non-steady-state approximation of Steele (39) during the OGTT as described elsewhere (34).

Rates of exogenous glucose appearance (RaE) and disappearance (RdE) were calculated from isotopic enrichment of blood glucose by 13C glucose, using the transposition of the Steele equation suggested by Proietto et al. (32)

R<SUB>aE</SUB> = <FR><NU>[R<SUB>aT</SUB> × (APE<SUB>2</SUB> + APE<SUB>1</SUB>)/2] + [<IT>p</IT>v(G<SUB>1</SUB> + G<SUB>2</SUB>)/2 × (APE<SUB>2</SUB> − APE<SUB>1</SUB>)/(<IT>t</IT><SUB>2</SUB> − <IT>t</IT><SUB>1</SUB>)]</NU><DE>APE<SUB>Glu,ing</SUB></DE></FR>


R<SUB>dE</SUB> = R<SUB>aE</SUB> − <FR><NU>[<IT>p</IT>v(G<SUB>1</SUB> + G<SUB>2</SUB>)/2 × (APE<SUB>2</SUB> − APE<SUB>1</SUB>)/(<IT>t</IT><SUB>2</SUB> − <IT>t</IT><SUB>1</SUB>)]</NU><DE>APE<SUB>Glu,ing</SUB></DE></FR>
where APE1 and APE2, correspond to the 13C APE at times t1 and t2, respectively, and G1 and G2 correspond to the plasma glucose levels at times t1 and t2. The pool fraction value (p) was taken as 0.75, and the distribution volume (v) was taken as 0.21/kg.

SGP was calculated as SGP = RaT - RaE.

Total lipid (LOx) and glucose oxidation (GOx) rates were estimated from gaseous exchanges measurements (14). Suprabasal glucose oxidation GOxSB was calculated as GOx during the OGTT - GOx before the OGTT (times -30 to 0). The exogenous glucose oxidation rate (Gexo, mg · kg-1 · min-1) was also calculated from 13C enrichment of expired CO2 as described by Normand et al. (30)
G<SUB>exo</SUB> = <FR><NU>∂<SUB>p</SUB>1,000 CO<SUB>2</SUB>(<IT>t</IT>) − ∂<SUB>p</SUB>1,000 CO<SUB>2</SUB>(<IT>t</IT><SUB>0</SUB>)</NU><DE>∂<SUB>p</SUB>1,000 Glu<SUB>ing</SUB> − ∂<SUB>p</SUB>1,000 CO<SUB>2</SUB>(<IT>t</IT><SUB>0</SUB>)</DE></FR>
× <FR><NU>V<SC>co</SC><SUB>2</SUB>(<IT>t</IT>) × 180,000</NU><DE>22.4 × 6 × body weight</DE></FR>
where partial p1,000 Gluing is the partial  value of ingested glucose measured after complete combustion in the elemental nitrogen and carbon analyzer on line with the isotope-ratio mass spectrometer; partial p1,000 CO2(t0) is the partial  value of the expired CO2 during times -30 to 0; partial p1,000 CO2(t) is the partial  value of the sampled CO2 at time t (end of each 30-min period); VCO2(t), expressed in liters per minute, is the mean volume of exhaled CO2 during periods of 30 min; 180,000 mg is the molecular weight of glucose; 1/22.4 is for the conversion into moles of CO2; and 1/6 is for the conversion into moles of glucose.

In four subjects, the effect of Ivélip infusion alone on 13C enrichment of exhaled CO2 was determined, and partial p1,000 CO2(t) of the OGTT during lipid infusion was corrected before calculation of a corrected Gexo: Gexo,corr, as follows
∂<SUB>p</SUB>1,000 CO<SUB>2</SUB>(<IT>t</IT>)<SUB>corr,Ivelip−OGTT</SUB> = ∂<SUB>p</SUB>1,000 CO<SUB>2</SUB>(<IT>t</IT>)<SUB>Ivelip−OGTT</SUB>
− [∂<SUB>p</SUB>1,000 CO<SUB>2</SUB>(<IT>t</IT>)<SUB>Ivelip,alone</SUB> − ∂<SUB>p</SUB>1,000 CO<SUB>2</SUB>(<IT>t</IT> − 120)<SUB>Ivelip,alone</SUB>]
Gexo,corr was then calculated by the same formula as Gexo, using values of partial p1,000 CO2(t)corr,Ivélip-OGTT instead of values of partial p1,000 CO2(t). This must be considered as a maximal correction for the contribution of 13C from the lipid infusion, since the infused lipids must be oxidized before their 13C appears in exhaled CO2, and such oxidation will tend to be reduced during the OGTT (38).

Glucose nonoxidative disposal was calculated using three different relationships
G<SUB>non-Ox1</SUB> = G<SUB>ingested</SUB> − G<SUB>Ox SB</SUB>
which is a balance between glucose input and glucose disappeared from the whole organism, as calculated in previous OGTTs under lipid infusions (26, 37)
G<SUB>non-Ox2</SUB> = R<SUB>dT</SUB> − G<SUB>Ox</SUB>
as proposed by Féry et al. (17), which represents nonoxidized glucose leaving plasma, and takes account of lipid-induced changes in glucose turnover rate
G<SUB>non-Ox3</SUB> = R<SUB>dE</SUB> − G<SUB>exo</SUB>
which represents exogenous glucose which had left plasma but did not contribute to exhaled CO2 and is not affected by lipid-induced alterations in gluconeogenesis, since it is not based on the indirect calorimetric equations. Gnon-Ox3,corr was also calculated, Gnon-Ox3,corr = RdE - Gexo,corr.

Turnover and oxidation rates values were calculated for 30-min intervals; cumulative values for the whole OGTT (330 min) are also presented.

Statistical analysis. Results are shown as means ± SE. Comparisons were performed by one-way ANOVA for repeated measurements followed by a t-test (within test comparison) and a t-test for nonpaired data (between test comparison). P < 0.05 was considered significant.

    RESULTS
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Methods
Results
Discussion
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Substrates and hormones. The data for substrates and hormones are presented in Figs. 2 and 3. The lipid infusion induced a continuous rise in plasma triglycerides and prevented the fall in FFA during the OGTT. Triglycerides and FFA were significantly higher from the beginning to the end of the lipid infusion, except at the last time point (+330 min), when FFA did not differ between the saline and Ivélip tests. Plasma glucose levels did not differ before the tests (times -120 and -90) and even before the oral load, after 90 min of lipid infusion. The lipid infusion induced a slight impairment in glucose tolerance, as shown by significantly higher values from times +120 to +180 (P < 0.05). Insulin and C-peptide levels were also slightly higher during the lipid infusion, with significant differences from times +180 to +240 (P < 0.05). The glycerol infusion had no significant effect on any substrate or hormone level, except on plasma glycerol, which was higher than during the saline infusion from time +120 to the end of the test (P < 0.05). However, this increase was less important than that observed during the lipid infusion (P < 0.05 from times -30 to +300).


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Fig. 2.   Time course of plasma glucose (mM), insulin (pM), and C-peptide (nM) before (times -120 to 0 min) and after (times 0 to 330 min) oral load. open circle , Controls; bullet , Ivélip tests; black-triangle, glycerol tests. * P < 0.05 between Ivélip and saline controls.


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Fig. 3.   Time course of plasma glycerol (µM), free fatty acid (FFA, µM), and triglycerides levels (mM) before (times -120 to 0 min) and after (times 0 to 330 min) oral load. open circle , Controls; bullet , Ivélip tests; black-triangle, glycerol tests. * P < 0.05 between Ivélip and saline controls.

Isotopic enrichments. Isotopic enrichment data are given in Fig. 4. D-[6,6-2H2]glucose enrichments declined comparably during the OGTT under saline, glycerol, and lipid infusion, reverting to postabsorptive values at the end of tests. However, as shown in Fig. 4, there was a trend for lower values under Ivélip. Before the oral load was ingested, no differences in the 13C level in glucose were detected among the saline, glycerol, and lipid infusion tests. It rose during the first 2 h of the OGTT and then declined without returning to baseline at the end of the tests. This decrease was slower during the lipid infusion, with significantly higher 13C enrichments of plasma glucose at times +180, 210, 240, 300 (P < 0.05), and 330 (P < 0.01). During glycerol infusion, 13C enrichments of plasma glucose were lower (P < 0.05 at times +60, 90, and 120). 13C enrichment of expired CO2 was significantly lower during the last 150 min of the Ivélip tests than during control tests (P < 0.01). However, the infusion of Ivélip alone in four subjects produced a progressive decline in 13C enrichment of expired CO2, with values from time +90 to the end of test below those at time -120 (P < 0.05). As shown in Fig. 4, no difference in 13C enrichment of expired CO2 was found between the Ivélip and saline tests after correction for the contribution of 13C from the infused lipids.


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Fig. 4.   Time course of isotopic enrichments of plasma glucose with D-[6,6-2H2]glucose (molar ratio × 100), plasma glucose with [13C] glucose [atom% excess (APE) × 1,000], and expired CO2 with 13C (partial p × 1,000) before (times -120 to 0 min) and after (times 0 to 330 min) oral load. open circle , Controls; bullet , Ivélip tests; black-triangle, glycerol tests; , Ivélip tests without oral glucose tolerance test; , partial p 1,000 CO2(t) corrected for contribution of 13C from Ivélip. * P < 0.05 between Ivélip and saline controls.

Rates of glucose appearance. Rates of glucose appearance are given in Table 1. Before the OGTT, postabsorptive SGP rates were identical during control (2.32 ± 0.11 mg · kg-1 · min-1), Ivélip (2.32 ± 0.05 mg · kg-1 · min-1), and glycerol (2.46 ± 0.18 mg · kg-1 · min-1) tests. Cumulative RaT over 330 min were higher during the lipid infusion and slightly increased during the glycerol infusion [saline 1,181 ± 71 mg/kg vs. Ivélip 1,388 ± 100 mg/kg (P < 0.05), glycerol 1,276 ± 126 mg/kg (NS vs. saline)]. Cumulative RaE over 330 min were higher during the lipid infusion and lower during the glycerol infusion [saline 864 ± 38 mg/kg vs. Ivélip 993 ± 67 mg/kg (P < 0.05), glycerol 698 ± 117 mg/kg (P < 0.05 vs. saline)]. Cumulative SGP over 330 min were higher during the lipid and glycerol infusions [saline 317 ± 57 mg/kg vs. Ivélip 395 ± 58 mg/kg (P < 0.05), glycerol 570 ± 54 mg/kg (P < 0.05 vs. Ivélip and P < 0.001 vs. saline)].

                              
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Table 1.   Glucose fluxes and oxidation during oral glucose tolerance test

Rates of glucose disappearance. Rates of glucose disappearance are given in Table 1. No glucose was detected in urine in any of the tests. Cumulative RdT over 330 min were higher during the lipid infusion (saline 1,242 ± 67 mg/kg vs. Ivélip 1,401 ± 96 mg/kg; P < 0.05). This was also observed for RdE but did not reach significance (saline 810 ± 52 mg/kg vs. Ivélip 885 ± 72 mg/kg; NS). Cumulative GOx over 330 min was lower during lipid infusion (saline 645 ± 19 mg/kg vs. Ivélip 405 ± 33 mg/kg; P < 0.001). This was also observed for Gexo (saline 423 ± 39 mg/kg vs. Ivélip 266 ± 60 mg/kg; P < 0.05), but the difference was smaller and was abolished by correction for the contribution of 13C from the lipids (Gexo,corr: 360 ± 92 mg/kg; NS vs. saline). Although different results were obtained from the three different relationships used to calculate nonoxidative glucose disposal, in every case the values were significantly higher with the lipid infusion (Gnon-Ox1: saline 666 ± 51 mg/kg vs. Ivélip 806 ± 28 mg/kg, P < 0.05; Gnon-Ox2: saline 597 ± 78 mg/kg vs. Ivélip 996 ± 0.95 mg/kg, P < 0.001; Gnon-Ox3: saline 392 ± 100 mg/kg vs. Ivélip 619 ± 95 mg/kg, P < 0.001). This was also true when Gexo,corr was used in place of Gexo (Gnon-Ox3,corr: 545 ± 83 mg/kg; P < 0.05 vs. saline).

    DISCUSSION
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Methods
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References

The present study was designed to shed more light on the mechanism of the moderate glucose intolerance induced by a lipid infusion during an OGTT. The results of our control OGTTs are in agreement with those of other studies using the same duration (5-6 h) and 1 g/kg oral glucose load (16, 27, 41). In line with Rousselle et al. (37) and Kruszynska et al. (23), we found that lipid infusion produced a moderate glucose intolerance. This was due to a higher rate of total glucose appearance, an impairment in the suppression of EGP, and an apparent higher appearance of exogenous glucose. The higher rate of total glucose appearance was partially reproducible by a glycerol infusion. The total glucose disappearance rate was also higher during lipid infusion despite a decrease in glucose oxidation, due to an increase in nonoxidative disposal. Total glucose oxidation, evaluated by indirect calorimetry, was more inhibited than exogenous glucose oxidation evaluated by the recovery of 13C in exhaled CO2. Although nonoxidative glucose disposal was only evaluated indirectly, the three different methods of calculation led to the same conclusion.

The slight defect in the suppression of SGP induced by the lipid infusion was observed despite slightly higher plasma levels of insulin and glucose, which are known to suppress SGP (11). This demonstrates that lipids can produce a state of hepatic resistance to insulin (24) but also resistance to the suppressive effect of glucose. This effect (395 - 317 approx  78 mg · kg-1 · 330 min-1) seems moderate, but it was probably underestimated. SGP was calculated by substracting RaE from RaT. We found an increased RaE under lipid infusion. This unexpected finding might reflect a higher absorption of glucose by the gut as proposed by Meylan et al. (26) but has yet to be demonstrated. Lipid infusion could also have, contrary to the total body glucose uptake, lowered the splanchnic uptake of the oral load. In fact, peripheral and splanchnic glucose uptake have been shown to be regulated differently (11). However, this would not explain why the lipid-induced increase in RaE only appeared at the end of the tests (times +150 to +240). In agreement with the results of Tissot et al. (41), calculated RaE remains positive 330 min after the oral load had been ingested in our control tests (0.65 ± 0.22 mg · kg-1 · min-1), when all metabolic parameters had returned to initial values. At this time, glucose is no longer absorbed (1), and the persistence of a significant enrichment in 13C of plasma glucose is an indication of recycling of the label through the gluconeogenic pathway. This rate of recycling has been estimated to be ~10% of the metabolism of the glucose load (15). It could have been increased by the lipid infusion, since FFA can stimulate of gluconeogenesis. Indeed Clore et al. (10) have demonstrated that the activity of the Cori cycle can be doubled by a lipid-heparin infusion in normal subjects. It is thus quite possible that an enhanced gluconeogenesis during lipid infusion led to an increased recycling of the 13C label, giving an erroneously high RaE and an underestimate of the calculated SGP (SGP = RaT - RaE).

Lipid emulsions contain free glycerol, which could contribute to the enhanced SGP. Had all this glycerol been used for gluconeogenesis, it would represent a production of 62 mg · kg-1 · 330 min-1 glucose, thus explaining the main part of the increased SGP (78 mg · kg-1 · 330 min-1). However, only 45-50% of infused glycerol is converted to glucose, even at high rates of glycerol infusion (31). It is unlikely that this proportion was increased by the presence of high FFA levels, since glycerol metabolism already generates NADH. To solve this issue, we performed glucose tolerance tests with glycerol infusion. As shown in Fig. 3, plasma glycerol rose only slowly compared with an almost square-wave rise in concentration during Ivélip infusion. Presumably, during Ivélip infusion, glycerol is released from the infused triglycerides by the action of lipoprotein lipase in peripheral tissues, which also explains the rise in FFA. A primed infusion of glycerol, with a higher infusion rate, would therefore be necessary to match the systemic glycerol concentrations seen during Ivélip infusion. However, important effects on glucose metabolism were observed during our glycerol infusion. We observed a moderate increase in glucose RaT; RaE was surprisingly low and therefore calculated SGP high. In fact, the increase in SGP compared with saline glucose tolerance tests cannot be accounted for by the infused glycerol. The 13C abundance in this glycerol was only slightly less than in the glycerol present in the lipid emulsion infused (-30.2 vs. -29.5 partial 13Cp × 1,000). Thus this cannot explain why 13C abundance in plasma glucose during the OGTT with glycerol infusion was so low. We think that, in the absence of high FFA levels, the recycling of [13C]glucose through gluconeogenesis from [13C]lactate was decreased by the glycerol infusion. This possibility is supported by previous demonstrations that glycerol infusion inhibits gluconeogenesis from other precursors (20, 40). Whatever the exact mechanism, this makes difficult to quantify accurately the contribution of glycerol to SGP. It is probable that the higher glucose RaT which we found during glycerol infusion reflects an enhanced SGP. We therefore think, in accordance with results from Boden and Chen (4), that the glycerol content of the lipid emulsion probably played a role in the modification of SGP.

RdT was not evaluated in previous studies on lipid-induced glucose intolerance (26, 37), except for the study of Kruszynska et al. (23). In agreement with this last study, we found that the lipid infusion increased glucose RdT during OGTT. This contrasts with results obtained during hyperinsulinemic euglycemic clamps (24). Using the glucose-clamp technique, Boden et al. (6) and Bonadonna et al. (7) have shown that a lipid infusion takes 3-4 h to inhibit glucose uptake. This may account for the failure to observe an inhibitory effect of lipids on glucose uptake in some studies but not in the present one, since we infused lipids for 7 h (beginning 90 min before glucose ingestion) and RdT was higher at the end of the Ivélip tests. Increased plasma insulin and glucose levels are recognized stimulators of peripheral glucose uptake (11). This may well account for the higher RdT we found: plasma insulin and glucose levels were significantly higher under lipid infusion in our study (which was not the case in clamp experiments). This higher RdT was observed despite a decrease in glucose oxidation as determined by indirect calorimetry, which is an early and consistent consequence of a lipid infusion (4, 6, 23, 24, 26, 37). Measurement of expired 13CO2 can also be used to measure glucose oxidation after an oral glucose load enriched with [13C]glucose. At first glance, both techniques gave the same result: the lipid infusion led to a ~40% inhibition in GOx and Gexo. However, the lipid infusion had a low abundance in 13C, which had its own effect on 13CO2 excretion rate independent of any lipid-glucose interaction, as we noted in the four subjects studied without the oral glucose load. Correction for this effect abolished the apparent inhibitory effect of the lipid infusion on Gexo. This subtraction of the effect of the lipid infusion alone on 13CO2 excretion is maximal estimate of the correction, since it does not take account the decrease of lipid oxidation due to enhanced glucose metabolism during the OGTT (38). However, our result suggests that the inhibitory effect of lipid infusion is more pronounced on total than on exogenous glucose oxidation. It may therefore specially affect the rate of glycogen oxidation. Because glucose oxidation is decreased or unchanged depending on the method used, the higher RdT during lipid infusion appears to derive from an increase in nonoxidative disposal of glucose. This increase was significant regardless of the method of calculation (Gnon-Ox1 approx  +20%, Gnon-Ox2 and Gnon-Ox3 approx  +40%). Because it does not take account of the higher RdT, the first method of calculation underestimates the effect of the lipid infusion; this explains why Gnon-Ox was not always significantly higher in other lipid-modified OGTTs (26, 37). Our result contrasts with reports that lipids inhibit glucose storage either evaluated indirectly, as we did, or more directly by measuring glycogen synthase activity on muscle biopsies (6) or by NMR (36). However, all these results were obtained during euglycemic clamp and not with a simultaneous increase in plasma glucose and insulin levels as in the present study. As discussed previously, the lack of inhibition of glucose nonoxidative disposal in our study cannot, contrary to other studies (23), be explained by an insufficient period of lipid infusion, since we infused lipids for 7 h. A glycogen-sparing effect of a lipid infusion has been described in animals (22) and suggested in humans (29). Our findings suggest that the slight lipid-induced increases in glucose and insulin levels divert glucose metabolism toward storage. This is consistent with the metabolic origin of insulin resistance proposed by Felber et al. (13).

It is of interest to compare the abnormalities in glucose tolerance induced by lipid infusion with those observed during starvation and in subjects with impaired glucose tolerance or NIDDM. Féry et al. (16) studied the mechanism of starvation diabetes by performing double-labeled OGTTs in normal subjects before and after 4 days of fast. The effect of starvation was similar to our observations with the lipid infusion, namely, higher residual SGP, lower glucose oxidation, and higher nonoxidative disposal. RaE was not affected by fasting, but the use of [1-14C]glucose enabled correction for recycling, which was indeed slightly higher during fasting. Similar results have been reported by Mitrakou et al. (27) in "spontaneous" impaired glucose tolerance. The fate (oxidative or not) of utilized glucose was not reported in that study, but Felber et al. (12), using indirect calorimetry, found lowered values for oxidation but not for glucose nonoxidative disposal in obese subjects with impaired glucose tolerance. The results obtained in patients with NIDDM were different. In all cases (17, 18, 28), RaT and SGP were found elevated (although Féry et al. observed this only in severely hyperglycemic patients; Ref. 17), but RdT was not higher (at least after correction for glycosuria; Ref. 18). Glucose oxidation was lower (17), but there was also an impaired glucose nonoxidative disposal, as shown by similar (18) or even lower (28) values despite the hyperglycemia. This last abnormality cannot be explained by the effect of lipids, at least during acute administration.

In summary, a lipid infusion led to a moderate impairment in glucose tolerance in normal subjects. This was due to a defect in both the suppression of systemic glucose production and the stimulation of glucose oxidation. Infused glycerol contributed to the effect on systemic glucose production. The impairment in glucose oxidation is in accordance with Randle hypothesis. But this defect in glucose oxidation did not decrease insulin stimulated glucose disposal as initially proposed by Randle et al. (33) In fact, glucose intolerance was moderate because of a concomitant increase in glucose nonoxidative disposal. A further defect, concerning glucose nonoxidative disposal, therefore appears to be required for the development of NIDDM.

    ACKNOWLEDGEMENTS

The authors acknowledge S. P. Jarman for translation.

    FOOTNOTES

This study was promoted by Institut National de la Santé et de la Recherche Médicale and supported by a grant from Association de Langue Française pour l'Etude du Diabete et des Maladies Métaboliques Institut Servier du Diabète and Comité Interprofessionnel de la Dinde Française.

Address for reprint requests: V. Rigalleau, Service de Nutrition et Diabétologie, USN, Hôpital Haut-Lévêque, Ave. de Magellan, 33600 Pessac, France.

Received 7 October 1997; accepted in final form 18 June 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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