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Am J Physiol Endocrinol Metab 292: E693-E701, 2007. First published October 24, 2006; doi:10.1152/ajpendo.00423.2006
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Mechanism of insulin-stimulated clearance of plasma nonesterified fatty acids in humans

André C. Carpentier,1 Frédérique Frisch,1 Pascal Brassard,1 François Lavoie,1 Annie Bourbonnais,1 Denis Cyr,2 Robert Giguère,2 and Jean-Patrice Baillargeon1

1Division of Endocrinology, Department of Medicine, and 2Department of Clinical Biochemistry, Centre Hospitalier Universitaire de Sherbrooke, Université de Sherbrooke, Québec, Canada

Submitted 17 August 2006 ; accepted in final form 23 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin increases plasma nonesterified fatty acid (NEFA) clearance in humans, but whether this is independent of change in plasma NEFA appearance is currently unknown. Nine nondiabetic men (age: 28 ± 3 yr, body mass index: 27.2 ± 1.7 kg/m2) underwent euglycemic clamps to maintain low (LINS) vs. high (HINS) physiological insulin levels for 6 h. An intravenous infusion of heparin + Intralipid (HI) was performed during 4 of the 6 h of the clamps (in the last 4 h at LINS and in the first 4 h at HINS), whereas saline infusion (SAL) was administered in the remaining 2 h to modulate plasma NEFA levels independently of plasma insulin levels. Four experimental conditions were obtained in each individual: LINS with saline (LINS/SAL) and with HI infusion (LINS/HI) and HINS with saline (HINS/SAL) and with HI infusion (HINS/HI). Plasma palmitate appearance during HINS/SAL was lower than during the three other experimental conditions (P < 0.05). In contrast, plasma linoleate appearance, as expected, was increased by HI independently of insulin level (P < 0.02). Plasma palmitate clearance during HINS/SAL was higher than LINS/SAL and LINS/HI (P < 0.008), and this increase was blunted during HINS/HI. We observed a linear decrease in plasma palmitate clearance with increasing plasma NEFA appearance independent of insulin levels. Plasma NEFA levels increased exponentially with increase in plasma NEFA appearance. We conclude that insulin stimulates plasma NEFA clearance by reducing the endogenous appearance rate of NEFA. The relationship between plasma NEFA level and appearance rate is nonlinear.

human; insulin action; lipid metabolism; fatty acid metabolism

UNDERSTANDING THE REGULATION of plasma nonesterified fatty acid (NEFA) is very important to elucidate its potential role in the development of various pathological conditions, including type 2 diabetes (27). Prolonged experimental elevation of plasma NEFA in humans reproduces the cardinal pathophysiological features of type 2 diabetes, including reduced insulin-mediated glucose utilization, impaired glucose-mediated insulin secretion, and increased endogenous glucose production (10, 36, 39). Epidemiological studies have linked elevated plasma NEFA levels to insulin resistance and type 2 diabetes (15, 26, 35), and reduced suppression of postprandial plasma NEFA concentration is among the earliest metabolic abnormalities seen in the natural history of type 2 diabetes (3).

Insulin potently suppresses plasma NEFA appearance. It is often assumed that plasma NEFA level is an accurate reflection of plasma NEFA appearance rate in various physiological and pathophysiological conditions known to affect plasma NEFA levels through change in plasma insulin level, i.e., that plasma NEFA clearance does not change with changing levels of plasma insulin. However, this may not necessarily be the case, because insulin has been shown to stimulate NEFA transport across plasma cell membranes (6). Elevation of plasma insulin level was associated with enhanced plasma NEFA clearance in human studies during the postabsorptive (32) and the postprandial state (31) and during enhanced intravascular triglyceride lipolysis using intravenous infusion of heparin plus Intralipid (9). In all of the latter in vivo studies, however, stimulation of plasma NEFA clearance by insulin did occur concomitantly with insulin-mediated suppression of plasma NEFA appearance rate and reduction in plasma NEFA levels. To our knowledge, whether plasma NEFA clearance can be saturated at high vs. low physiological NEFA levels has not been formally established in vivo in humans. Thus it is unclear from previous in vivo studies whether insulin-stimulated clearance of plasma NEFA may be a direct effect or whether it is secondary to change in plasma NEFA levels or appearance.

The aim of the present study was to determine by what mechanism insulin stimulates plasma NEFA clearance rate in vivo in humans. Our hypothesis was that insulin may stimulate clearance of plasma NEFA independently of its effect on plasma NEFA appearance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Nine nonsmoking, healthy Caucasian men (mean body mass index: 27.2 ± 1.7 kg/m2) ages 21 to 50 yr (mean age: 28 ± 3 yr) participated in the present study (Table 1). None had diabetes based on repeated assessment of fasting glucose concentration and 75-g oral glucose tolerance test (1). None were taking any medication, had any current medical condition known to affect lipid levels or insulin sensitivity, or had known cardiovascular disease. Informed, written consent was obtained from all participants in accordance with the Declaration of Helsinki, and the study was approved by the Human Ethics Committee of the Centre Hospitalier Universitaire de Sherbrooke.


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  		Table 1. Characteristics of the study participants

 
Experimental protocols. All subjects participated in two studies 3–4 wk apart in random order. They avoided changes in lifestyle and weight throughout the study. They were told to avoid strenuous exercise for 48 h before each study and were provided with dietary instructions to maintain a eucaloric diet prescribed by a registered dietician, following a 3-day food diary. Compliance was ascertained on the morning of each study. The subjects were admitted at our metabolic investigation center on each occasion between 7:30 and 8:30 AM following a 12-h overnight fast and remained fasting for the duration of the study. On arrival, body weight and height were measured, and lean body mass was determined using electrical bioimpedance (Hydra ECF/ICF; Xitron Technologies, San Diego, CA). An intravenous catheter (IV) was placed in one forearm for infusions, and another IV was placed in a retrograde fashion in the contralateral arm, maintained in a heating box (~55°C) for blood sampling.

The protocols (Fig. 1) were designed to determine plasma NEFA kinetics at low peripheral insulin level (peripheral plasma insulin level was similar to that during fasting, but this condition was not identical to the fasting condition because insulin was not replaced in the portal circulation; protocol A) vs. high insulin level (protocol B) with an intravenous infusion of heparin (250 U/h) and 20% Intralipid (40 ml/h; a triglyceride emulsion composed of 50% linoleate, 26.5% oleate, 10.5% palmitate, 8.5% linolenate, and 3.5% stearate) (11) vs. saline intravenous infusion to change plasma NEFA levels and appearance rate independently of plasma insulin levels. Heparin + Intralipid (HI) intravenous infusion was administered during 4 of the 6 h in both protocols (between 120 and 360 min in protocol A and between 0 and 240 min in protocol B). We selected these timings and orders of infusion because we had previously shown that insulin stimulates plasma palmitate clearance during intravenous administration of HI for 4 h (9) and because 2 h allows sufficient time for measurement of large changes of plasma NEFA kinetics with or without insulin in the absence of administration of HI (32). From these previous studies, we could predict changes in plasma palmitate and glycerol kinetics from saline infusion to HI infusion at low insulin level, but not the reverse. These studies also allowed us to estimate changes in plasma palmitate and glycerol kinetics from baseline during simultaneous infusion of high insulin and HI and once the HI infusion was stopped while maintaining high plasma insulin level. We had no previous data to predict changes in plasma NEFA kinetics from saline infusion to HI infusion while maintaining high plasma insulin level. Low plasma insulin was maintained by a continuous low (0.05 mU·kg–1·min–1) insulin infusion (Novolin R; NovoNordisk) during the 6 h of protocol A, whereas high insulin was obtained using a primed (0.8 mU/kg) continuous high (1.2 mU·kg–1·min–1) insulin infusion (with KCl at 10 meq/h to avoid insulin-induced hypokalemia) in protocol B. A variable infusion of dextrose (20%) was adjusted to maintain plasma glucose at ~5.5 mmol/l throughout both protocols. Octreotide (30 µg/h Sandostatin; Sandoz) and human growth hormone (GH; 3 ng·kg–1·min–1 Nutropin; Roche) were administered in the two protocols (23). Glucagon was not replaced, because it can result in insulin secretion breakthrough at low insulin infusion rate (23) and because it has minimal effect on NEFA metabolism in humans (4, 21, 33).


Figure 1
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  		Fig. 1. Experimental protocols. All of the participants underwent 2 euglycemic clamps with octreotide + growth hormone (GH) intravenous (IV) infusion to maintain low (protocol A) or high (protocol B) plasma insulin level for 6 h. IV infusions of 20% dextrose (D20%) and KCl were administered to maintain plasma glucose at fasting level and to avoid a decrease in serum potassium level induced by insulin. In protocol A, an intravenous infusion of saline was administered between 0 and 120 min, followed by an IV infusion of heparin + Intralipid between 120 and 360 min. In protocol B, an IV infusion of heparin + Intralipid was administered between 0 and 240 min, followed by an IV infusion of saline between 240 and 360 min. In both protocols, a primed continuous IV infusion of [1,1,2,3,3-2H5]glycerol and a continuous IV infusion of [U-13C]palmitate and [9,10,12,13-3H]linoleate were administered from 0 to 360 min.

 
An intravenous infusion of [U-13C]potassium palmitate (in 100 ml of 5% human serum albumin; Cambridge Isotopes Laboratories, Andover, MA) and a primed (1.6 µmol/kg) continuous infusion of [1,1,2,3,3-2H5]glycerol (Cambridge Isotopes Laboratories) were administered through a 0.22-µm Millipore filter for the duration of the study to quantify plasma palmitate and glycerol turnover, as described previously (9). In addition, an intravenous infusion of [9,10,12,13-3H]linoleate (American Radiolabeled Chemical) was administered throughout both protocols. The rate of infusion of these tracers was modified during the different phases of the study to minimize change in plasma tracer enrichment due to expected changes in appearance rate (32). We started from the following infusion rates at low insulin level with saline infusion (LINS/SAL): 0.01 µmol·kg–1·min–1, 0.05 µmol·kg–1·min–1, and 0.15 µCi/min for [U-13C]palmitate, [1,1,2,3,3-2H5]glycerol, and [9,10,12,13-3H]linoleate, respectively. We multiplied these rates of infusion by 2, 2.5, and 4 during low insulin with HI infusion, by 1, 2.5, and 2 during high insulin with HI infusion, and by 0.5, 0.5, and 0.5 during high insulin with saline infusion. The assumptions made for these changes in infusion rates included 1) HI infusion at low plasma insulin level results in ~2-, 2.5-, and 4-fold elevation in plasma palmitate, glycerol, and linoleate appearance rates compared with saline infusion at low plasma insulin level (9); 2) HI infusion at high plasma insulin level results in approximately no change in plasma palmitate appearance and in 2.5- and 2-fold elevations in plasma glycerol and linoleate appearance rate compared with saline infusion at low plasma insulin level (9); and 3) high plasma insulin level during saline infusion results in ~2-fold reduction in plasma palmitate, glycerol, and linoleate appearance rates compared with low insulin level (32). In protocol A, the rates of [U-13C]palmitate, [1,1,2,3,3-2H5]glycerol, and [9,10,12,13-3H]linoleate appearance were, respectively, 1.21 ± 0.20 µmol/min, 8.25 ± 0.77 µmol/min, and 2.52 ± 0.39 x 105 dpm/min between 0 and 120 min and 2.14 ± 0.32 µmol/min, 19.66 ± 1.79 µmol/min, and 10.07 ± 1.55 x 105 dpm/min between 120 and 360 min. In protocol B, the rates of [U-13C]palmitate, [1,1,2,3,3-2H5]glycerol, and [9,10,12,13-3H]linoleate appearance were, respectively, 1.12 ± 0.19 µmol/min, 21.75 ± 3.04 µmol/min, and 5.26 ± 0.73 x 105 dpm/min between 0 and 240 min and 0.56 ± 0.09 µmol/min, 4.52 ± 0.62 µmol/min, and 1.03 ± 0.19 x 105 dpm/min between 240 and 360 min. All tracers were tested for sterility and pyrogenicity before use.

After a 30-min bed rest, blood samples were taken at 10-min intervals at baseline (–20 to 0 min) and between 100 and 120 min, 220 and 240 min, and 340 and 360 min. Blood was collected in tubes containing Na2EDTA and Orlistat (30 µg/ml: Roche, Mississauga, Ontario, Canada) to prevent in vitro triglyceride lipolysis.

Laboratory assays. Glucose was assayed at bedside (Beckman Glucose Analyzer II; Beckman Instruments, Fullerton, CA). Insulin, glucagon, and GH were measured by specific radioimmunoassays (Linco, St. Charles, MO, and Nichols Institute Diagnostics, San Juan Capistrano, CA). Total plasma NEFA and triglycerides were measured using colorimetric assays (Wako Industrials and Thermo DMA, respectively). Plasma glycerol was extracted and derivatized with bis(trimethylsilyl)trifluoroacetamide + 10% trimethylchlorosilane (Regis Technologies, Morton Grave, IL), and plasma glycerol and [1,1,2,3,3-2H5]glycerol enrichment were measured by GC-MS as previously described (9). Plasma palmitate, linoleate, oleate, and [U-13C]palmitate enrichment were measured by LC-MS as previously described (9). Another aliquot of the supernatant obtained after extraction of plasma NEFA was counted with a LS6500 Multipurpose scintillation counter (Beckman Coulter) after evaporation of its water content to quantify plasma [3H]linoleate activity.

Calculations. Plasma palmitate appearance rate (RaP) was calculated from the C16:0 M+16 enrichment of plasma palmitate from background and tracer infusion rate (TTRP) as described previously (5). Plasma linoleate appearance rate (RaL) was also calculated from plasma [9,10,12,13-3H]linoleate specific activity (in dpm/µmol; SAL) using Steele's steady-state equation (40). Total plasma NEFA appearance rate (RaNEFA) was determined using the following calculation (24): RaNEFA = (RaP + RaL) x {[NEFA]/([palmitate] + [linoleate])}, where [NEFA], [palmitate], and [linoleate] are plasma NEFA, palmitate, and linoleate concentrations, respectively. This approach assumes that plasma oleate kinetics (the other prevalent NEFA in plasma) can be estimated from plasma palmitate and linoleate kinetics (22, 32, 38). Plasma glycerol appearance rate (RaGLYC) was also determined from plasma glycerol tracer (M+5) enrichment from baseline (TTRGLYC) and the tracer infusion rate (25). Clearance rates of plasma palmitate, linoleate, NEFA, and glycerol (CLP, CLL, CLNEFA, and CLGLYC, respectively) were determined by dividing their respective appearance rates by their plasma concentrations. Appearance rate of plasma NEFA reflects the net production of plasma NEFA from both intracellular and extracellular lipolysis of complex lipids (in particular triglycerides) plus any direct production of circulating NEFA from de novo lipogenesis. Clearance of plasma NEFA reflects the net volume of plasma that looses its content of NEFA per amount of time and is theoretically stimulated by increased net intracellular uptake and/or utilization of NEFA (oxidation or esterification in complex lipids, including assembly of lipoproteins).

Statistical analyses. Data are expressed as means ± SE. Data at baseline and between 100 to 120, 220 to 240, and 340 to 360 min were averaged and compared using ANOVA for repeated measures with Scheffé's test for multiple comparisons. Because HI infusion for 2 vs. 4 h did not affect the levels or kinetics of plasma NEFAs and glycerol and did not affect plasma insulin levels, data between 220 to 240 min and 340 to 360 min in protocol A and between 100 to 120 min and 220 to 240 min in protocol B were averaged for statistical analyses. Thus four experimental conditions in which NEFA and glycerol kinetics were assessed could be compared within each subject: 1) LINS/SAL, 2) low insulin level with HI intravenous infusion (LINS/HI), 3) high plasma insulin level with saline intravenous infusion (HINS/SAL), and 4) high plasma insulin level with HI intravenous infusion (HINS/HI). All metabolic parameters measured during these four experimental conditions were examined using an ANOVA for repeated measures and Scheffé's test for multiple comparisons. The relationship among plasma NEFA clearance, appearance rate, and level during the four experimental conditions was assessed using the Marquardt-Levenberg algorithm (SigmaPlot 2001 for Windows, version 7.0; SPSS). The type of function (linear or exponential rise) that best described the relationship between two variables was determined from the maximal R2 value of the model. For all analyses, a two-tailed P value <0.05 was considered significant. Statistical analyses were performed using the SAS software for Windows (version 9.1; SAS Institute, Cary, NC).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Plasma glucose, insulin, GH, glucagon, total NEFA, and triglycerides at baseline and during glucose clamps at low and high insulin level. The mean baseline glucose, insulin, GH, glucagon, total NEFA, and triglyceride levels were 4.9 ± 0.1 mmol/l, 75 ± 15 pmol/l, 0.48 ± 0.19 µg/l, 81 ± 7 ng/l, 488 ± 54 µmol/l, and 1.56 ± 0.78 mmol/l, respectively. By design, plasma glucose levels were not significantly different throughout both protocols and were matched between the four experimental conditions (LINS/SAL, LINS/HI, HINS/SAL, and HINS/HI) (Fig. 2A). Plasma insulin levels were significantly increased from baseline during HINS/SAL and HINS/HI (P < 0.001) but did not change during LINS/SAL and LINS/HI (Fig. 2B). Plasma GH and triglyceride levels were not significantly different from baseline in the four experimental conditions (not shown). Plasma glucagon levels were significantly reduced compared with baseline (P < 0.001) but were not different in the four experimental conditions (not shown). Total plasma NEFA levels (Fig. 2C) did not change from baseline during LINS/SAL but were significantly increased by approximately twofold during LINS/HI (P < 0.001). Total plasma NEFA levels did not significantly change from baseline during HINS/HI (0–240 min) but were significantly reduced during HINS/SAL (P < 0.001).


Figure 2
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  		Fig. 2. Plasma glucose (A), insulin (B), and total nonesterified fatty acid (NEFA) levels (C) during euglycemic clamp at low (fasting; bullet) vs. high (hyperinsulinemia; {circ}) plasma insulin level.

 
Concentration of plasma palmitate, oleate, linoleate, and glycerol and TTRP, [9,10,12,13-3H]linoleate specific activity, and TTRGLYC at baseline and during glucose clamps at low and high insulin level. The mean baseline plasma palmitate, oleate, linoleate, and glycerol levels were 128 ± 10, 204 ± 21, 100 ± 10, and 63 ± 5 µmol/l, respectively. Plasma palmitate levels (Fig. 3A) did not significantly change from baseline during LINS/SAL but were increased during LINS/HI between 220 and 240 min (P = 0.06 vs. baseline) and between 340 and 360 min (P < 0.03 vs. baseline). Plasma palmitate levels were not significantly changed from baseline during HINS/HI but were significantly reduced during HINS/SAL (P < 0.008). Trends similar to those for palmitate were observed with plasma oleate levels (ANOVA, P < 0.001) at low insulin level (208 ± 19, 231 ± 26, 288 ± 25, and 296 ± 30 µmol/l at baseline, 100 to 120 min, 220 to 240 min, and 340 to 360 min, respectively), although the differences between individual intervals did not reach statistical significance using Scheffé's test. Plasma oleate levels tend to decrease from baseline during HINS/HI and were significantly reduced during HINS/SAL (208 ± 29, 140 ± 14, 129 ± 16, and 24 ± 13 µmol/l at baseline, 100 to 120 min, 220 to 240 min, and 340 to 360 min, respectively, P < 0.001 for difference between the later interval vs. baseline). Plasma linoleate levels (Fig. 3B) did not significantly change from baseline during LINS/SAL but were increased approximately fourfold during LINS/HI (P < 0.001). Plasma linoleate levels increased significantly from baseline ~2.5-fold during HINS/HI between 100 and 120 min (P = 0.04) and between 220 and 240 min (P = 0.02) but were reduced below baseline level during HINS/SAL between 340 and 360 min (P < 0.001 vs. HI infusion). Although TTRP (Fig. 3C) tended to increase during LINS/HI compared with LINS/SAL and during HINS/SAL compared with HINS/HI, these differences were not statistically significant. TTRP was also similar at fasting and high plasma insulin level. [9,10,12,13-3H]linoleate specific activity (SAL; Fig. 3D) was not significantly different between LINS/HI and LINS/SAL. In contrast, SAL was significantly increased during HINS/SAL compared with HINS/HI (P < 0.001). Glycerol levels (Fig. 3E) were significantly increased from baseline during LINS/HI (P < 0.001) and during HINS/HI (P < 0.001). TTRGLYC (Fig. 3F) did not significantly change at fasting or during the hyperinsulinemic clamp.


Figure 3
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  		Fig. 3. Plasma palmitate (A) and linoleate levels (B), plasma palmitate tracer-to-tracee ratio (TTR; C), plasma [9,10,12,13-3H]linoleate specific activity (SA; D) during euglycemic clamp at low (bullet) vs. high ({circ}) plasma insulin level.

 
Plasma glucose, insulin, glycerol, and NEFA levels during LINS/SAL, LINS/HI, HINS/SAL, and HINS/HI. Plasma glucose levels were not significantly different among the four experimental conditions (Table 2). Plasma insulin levels were significantly higher during HINS/SAL and HINS/HI compared with LINS/SAL and LINS/HI (P < 0.001), whereas there was no significant difference between HI and SAL. Plasma glycerol levels were significantly higher during LINS/HI and HINS/HI than during LINS/SAL and HINS/SAL (P < 0.001) and were lower during HINS/SAL compared with LINS/SAL (P < 0.001). Plasma palmitate levels were significantly different among the four experimental conditions (P < 0.02), being the highest during LINS/HI, followed by LINS/SAL, then HINS/HI, and finally HINS/SAL. Plasma oleate levels displayed similar trends, although the difference between LINS/HI and LINS/SAL did not reach statistical significance. In contrast, plasma linoleate levels were highest during LINS/HI, followed by HINS/HI, then LINS/SAL, and finally HINS/SAL (P < 0.003 for difference among all conditions except the later two). Total plasma NEFA levels were similar between LINS/SAL and HINS/HI, whereas they were significantly higher during LINS/HI (P < 0.001 vs. all other conditions) and significantly lower during HINS/SAL (P < 0.001 vs. all other conditions).


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  		Table 2. Plasma glucose, insulin, glycerol, and NEFA levels during LINS/SAL, LINS/HI, HINS/SAL, and HINS/HI

 
Plasma glycerol and NEFA appearance and clearance rates during LINS/SAL, LINS/HI, HINS/SAL, and HINS/HI. RaGLYC was significantly higher during LINS/HI and HINS/HI compared with LINS/SAL and HINS/SAL (P < 0.002), but CLGLYC was not different among the four experimental conditions (Table 3). RaP was significantly lower during HINS/SAL compared with the other experimental conditions (P < 0.05). CLP showed opposite trends and was significantly higher during HINS/SAL compared with all other conditions (P < 0.008). RaL was significantly lower during HINS/SAL compared with LINS/HI and HINS/HI (P < 0.001), whereas it was intermediate during LINS/SAL (P < 0.02 vs. LINS/HI). CLL was also the lowest during HINS/SAL, followed by LINS/HI, then HINS/HI, and was highest during LINS/SAL (P < 0.02 for difference between HINS/SAL and LINS/SAL). RaNEFA was also significantly lower during HINS/SAL compared with the other three experimental conditions (P < 0.001). In contrast, CLNEFA was highest during HINS/SAL, followed by LINS/SAL, then HINS/HI, and finally LINS/HI (P < 0.004 for difference between HINS/SAL and LINS/HI).


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  		Table 3. Plasma glycerol and NEFA appearance and clearance rates during LINS/SAL, LINS/HI, HINS/SAL, and HINS/HI

 
Relationship among NEFA level, appearance rate, and clearance and plasma insulin level. CLP was linearly and inversely related to total plasma NEFA levels (R2 = 0.83, P = 0.06) and RaNEFA (R2 = 0.99, P = 0.002) (Fig. 4, A and B) but was not related to plasma insulin levels (Fig. 4C). CLL was not related to total NEFA levels, RaNEFA, or plasma insulin levels (Fig. 4, B, D, and F, respectively). Plasma palmitate, linoleate, and total NEFA levels increased exponentially with their respective appearance rates (Fig. 5) (R2 = 0.96, P = 0.01; R2 = 0.98, P = 0.007; and R2 = 0.94, P = 0.02 for palmitate, linoleate, and total plasma NEFA, respectively). CLGLYC was not associated with RaGLYC (Fig. 6A) or plasma insulin level (Fig. 6B), whereas plasma glycerol levels increased as a linear function of RaGLYC (Fig. 6C) (R2 = 0.96, P = 0.01).


Figure 4
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  		Fig. 4. Relationship between plasma palmitate clearance (CLP) and total plasma NEFA level (A), total plasma NEFA appearance rate (RaNEFA; B), or plasma insulin level (C) and relationship between plasma linoleate clearance (CLL) and total plasma NEFA level (D), RaNEFA (E), or plasma insulin level (F).

 

Figure 5
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  		Fig. 5. Relationship between plasma palmitate level and palmitate appearance rate (RaP; A), between plasma linoleate level and linoleate appearance rate (RaL; B), and between total plasma NEFA level and RaNEFA (C).

 

Figure 6
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  		Fig. 6. Relationship between plasma glycerol clearance (ClGLYC) and plasma glycerol appearance rate (RaGLYC; A) or plasma insulin level (B) and between plasma glycerol levels and RaGLYC (C).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have shown that preventing insulin-mediated suppression of plasma NEFA appearance rate blunts insulin-stimulated plasma palmitate clearance rate. Our results also show that plasma palmitate clearance is directly and inversely proportional to plasma NEFA appearance, independent of plasma insulin levels. The main mechanism by which insulin stimulates plasma palmitate clearance, as observed in previous human studies (9, 31, 32), is therefore through suppression of endogenous plasma NEFA appearance. Direct stimulation by insulin of NEFA transport across plasma membranes as demonstrated ex vivo (6) therefore does not appear to influence plasma NEFA clearance at the whole body level. This is further supported by the lower, not higher, plasma linoleate clearance at high vs. low insulin level during saline infusion.

The difference in behavior between palmitate and linoleate clearance with increasing plasma NEFA appearance could suggest different regulation of circulating palmitate and linoleate clearance. However, we were unable to avoid an increase in linoleate tracer specific activity during the hyperinsulinemic clamp with saline infusion (HINS/SAL). This could have led to an overestimation of plasma linoleate appearance (19, 20), leading to underestimation of plasma linoleate clearance during this experimental phase. The very important fall in plasma linoleate levels during HINS/SAL nevertheless suggests that plasma linoleate appearance was indeed much lower than during low insulin condition (LINS/SAL). Assuming an average reduction of plasma linoleate appearance from low insulin level of threefold during HINS/SAL (as observed for palmitate), the average clearance of plasma linoleate would have been estimated at ~1.47 l/min, a level compatible with an inverse relationship between plasma linoleate clearance and plasma NEFA appearance (see Fig. 4E). Moreover, the exponential growth relationship between plasma linoleate levels compared with appearance would still be present if plasma linoleate appearance during HINS/SAL (the lowest point on the curve) had been increased severalfold compared with the level reported (Fig. 5B), suggesting that linoleate clearance decreases with increasing appearance. Linoleate tracer infusion will need to be reduced further in future studies performed under similar conditions to address this important limitation of the present study regarding plasma linoleate kinetics. Until then, plasma linoleate kinetics during HINS/SAL in the present study should be regarded with caution.

Most of plasma linoleate appearance during intravenous HI infusion likely originated from intravascular lipolysis (~50% of fatty acids contained in Intralipid are linoleate), whereas most palmitate appearance probably derived from intracellular lipolysis (~10% of fatty acids contained in Intralipid). This limitation would also apply to glycerol, also expected to derive mostly from the intravascular compartment during HI infusion. In the case of glycerol, however, our results do not show lower clearance with higher appearance. In vivo experimental data in animals have suggested that circulating triglycerides (via intravascular lipolysis) and injection of heparin do not interfere with tissue uptake of plasma NEFA (2). Thus the use of heparin and Intralipid is unlikely to have interfered with interpretation of plasma NEFA clearance in the present study. Plasma NEFA tracer recirculation from incorporation into endogenous very low-density lipoprotein triglycerides is another potential source of error in the determination of true plasma NEFA appearance when using these methodologies. However, insulin did not change the specific activity of plasma triglycerides during intravenous infusion of radioactive oleate tracer in humans (37). These potential limitations of our experimental design are therefore unlikely to have affected our conclusions. Finally, we cannot exclude the possibility that octreotide infusion, fixed levels of GH, and/or low levels of glucagon as per our experimental protocols may have affected the conclusion of the present study with regard to the mechanism of insulin-stimulated clearance of plasma NEFA. However, we are not aware of evidence from previous studies that such could be the case.

To our knowledge, the present study is the first designed to determine the potential mechanism of stimulation of plasma NEFA clearance by insulin in vivo in humans. In an elegant series of in vitro and ex vivo experiments, Bonen and colleagues (7, 13, 14, 18, 29) have demonstrated that insulin acutely increases NEFA transport inside cardiac and skeletal muscle cells through stimulation of expression and translocation of CD36 to the plasma membrane independent of intracellular metabolism of fatty acids. A direct acute effect of insulin on transport of NEFA into tissues has been demonstrated in some (34) but not other (42) in vivo studies in animals. Although we have shown that insulin per se does not stimulate plasma NEFA clearance at the whole body level, our study cannot exclude a possible organ-specific stimulation of NEFA uptake by insulin with reduction in others. Moreover, it is possible that insulin channels NEFA flux preferentially through some metabolic pathways without changing net intracellular NEFA uptake as we recently showed in vivo in rats (16). Thus insulin may regulate intracellular transport of NEFA at least in some organs in vivo without apparent change in NEFA clearance at the whole body level.

The inverse relationship between plasma NEFA appearance and clearance implies that plasma NEFA levels increase disproportionately with increasing plasma NEFA appearance rate. Indeed, we have shown that plasma palmitate, linoleate, and NEFA levels rise as an exponential growth function of their appearance rates. This observation suggests that plasma NEFA level does not accurately reflect NEFA flux, i.e., that total tissue exposure to NEFA over time may be overestimated at higher levels, such as during fasting, and underestimated at lower levels, such as during the postprandial state, when judged by relative plasma NEFA concentration alone. The results of our study demonstrate saturation of clearance of plasma NEFA with increased appearance rate of NEFA. This raises the possibility that high plasma NEFA levels observed in subjects with insulin resistance may display abnormal regulation of plasma NEFA clearance in addition to increased plasma NEFA appearance. Interestingly, the insulin-sensitizer pioglitazone lowers plasma NEFA concentration by increasing plasma NEFA clearance rate independently of change in plasma NEFA appearance in nondiabetic individuals with abdominal obesity (37).

None of the participants in the present study had impaired glucose tolerance or type 2 diabetes, although one of the subjects (subject 6, see Table 1) was much heavier than the other participants. Removing this subject from the analysis did not affect our conclusion that insulin did not change NEFA clearance independently of change in plasma NEFA appearance. A previous study in subjects with uncontrolled type 2 diabetes has shown a reduced plasma NEFA clearance rate that was reverted toward normal with better control of hyperglycemia (41). Renal NEFA uptake also was shown to be reduced in patients with type 2 diabetes proportionally to enhanced renal glucose uptake (30). In the present study, glycemia did not correlate with plasma NEFA clearance (not shown). On the other hand, ex vivo studies have provided support for enhanced intramyocellular transport of plasma NEFA in patients with type 2 diabetes through increased expression of CD36 (8). Enhanced CD36-mediated transport of NEFA also was shown in red skeletal muscle and myocardium in obese rats with or without diabetes (12, 17, 28). Further studies are needed to determine the relation between plasma NEFA appearance and clearance in patients with disorders of glucose metabolism.

In conclusion, the present study demonstrates that insulin stimulates clearance of plasma NEFA in humans mainly through reduction of plasma NEFA appearance. The inverse relationship between plasma NEFA clearance and appearance explains the exponential increase of plasma NEFA levels with increase in plasma NEFA appearance in healthy men.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by Canadian Institutes of Health Research (CIHR) Grant MOP53094. A. C. Carpentier is a new investigator of the CIHR, and J. P. Baillargeon is a research scholar from the Fonds de la Recherche en Santé du Québec. P. Brassard is the recipient of a Fellowship Award from the Department of Medicine of the Université de Sherbrooke.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Carpentier, Division of Endocrinology, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4 (e-mail: andre.carpentier{at}usherbrooke.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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