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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 5 1834-1842
Copyright © 2007 by The Endocrine Society

Dose-Response Effects of Free Fatty Acids on Glucose and Lipid Metabolism during Somatostatin Blockade of Growth Hormone and Insulin in Humans

L. C. Gormsen, N. Jessen, J. Gjedsted, S. Gjedde, H. Nørrelund, S. Lund, J. S. Christiansen, S. Nielsen, O. Schmitz and N. Møller

Medical Department M (Endocrinology and Diabetes) (L.C.G., N.J., S.G., H.N., S.L., J.S.C., S.N., N.M.), Department of Anesthesiology (J.G.), and Immunoendocrine Research Unit (J.G.), Aarhus University Hospital, and Department of Pharmacology (O.S.), Aarhus University, DK-8000 Aarhus, Denmark

Address all correspondence and requests for reprints to: Lars C. Gormsen, M.D., Medical Department M, Aarhus University, Hospital, Nørrebrogade 42, DK-8000 Aarhus C, Denmark. E-mail: lars.christian.gormsen{at}ki.au.dk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: GH and other stress hormones stimulate lipolysis, which may result in free fatty acid (FFA)-mediated insulin resistance. However, there are also indications that FFAs in the very low physiological range have the same effect.

Objective: The objective of the study was to address systematically the dose-response relations between FFAs and insulin sensitivity.

Design: We therefore examined eight healthy men for 8 h (6 h basal and 2 h glucose clamp) on four occasions.

Intervention: Intralipid was infused at varying rates (0, 3, 6, 12 µl·kg–1·min–1); lipolysis was blocked by acipimox; and endogenous GH, insulin, and glucagon secretion was blocked by somatostatin and subsequently replaced at fixed rates.

Results: This resulted in four different FFA levels between 50 and 2000 µmol/liter, with comparable levels of insulin and counterregulatory hormones. Both in the basal state and during insulin stimulation, we saw progressively decreased glucose disposal, nonoxidative glucose disposal, and forearm muscle glucose uptake at FFA levels above 500 µmol/liter. Apart from forearm glucose uptake, the very same parameters were decreased at low FFA levels (~50 µmol/liter). FFA rate of disposal was linearly related to the level of FFAs, whereas lipid oxidation reached a maximum at FFA levels approximately 1000 µmol/liter.

Conclusion: In the presence of comparable levels of all major metabolic hormones, insulin sensitivity peaks at physiological levels of FFAs with a gradual decrease at elevated as well as suppressed FFA concentrations. These data constitute comprehensive dose-response curves for FFAs in the full physiological range from close to zero to above 2000 µmol/liter.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IN GENERAL, GH AND OTHER stress hormones acutely increase lipolysis and cause elevated free fatty acid (FFA) levels. This FFA-releasing effect of GH has been convincingly demonstrated during stress conditions such as exercise (1) and fasting (2) and in patients with acromegaly (3), although recent evidence indicates that long-term GH therapy may have more complex effects on lipolysis in, for example, lipodystrophic HIV patients (4). Elevated FFAs have beneficial protein conserving effects (5) but also induce insulin resistance (6) and contribute to GH-induced insulin resistance (7). When experimentally elevated, FFAs in the range between 300 and 1700 µmol/liter cause inhibition of insulin-stimulated glucose disposal and insulin signaling in muscle (8). The intracellular mechanism behind this FFA-induced insulin resistance may involve direct substrate competition; accumulation of citrate and glucose-6-phosphate, leading to enzyme inhibition (9); or accumulation of fatty acyl CoA, diacylglycerol inhibiting proximal insulin signaling, and glucose transporter-4 translocation (6, 10, 11). Lipid metabolites may also induce insulin resistance through inhibition of insulin signaling by activation of cytokines and inflammatory markers (12).

Despite clear evidence that FFAs cause insulin resistance, there is to our knowledge a lack of regular dose-response studies of the effects of FFAs on glucose and lipid metabolism, and little is known about FFA concentrations in the low physiological range (<500 µmol/liter). In a preliminary study, we observed that during blockade of lipolysis with acipimox an intralipid infusion increased both circulating FFA concentrations and, unexpectedly, also insulin sensitivity.

The aim of this study was therefore to define comprehensive dose-response effects of FFA on basal and insulin stimulated glucose and lipid metabolism in the full physiological range between approximately 50 and approximately 2000 µmol/liter. We hypothesized that: 1) FFA levels in the very low physiological range would cause insulin resistance, 2) higher FFA levels would inhibit insulin-stimulated glucose metabolism in a linear fashion, and 3) there would be a linear relation between FFA levels and FFA disposal and oxidation. To avoid FFA-related changes in insulin (13) and GH (14), we infused somatostatin and blocked endogenous lipolysis by acipimox and studied eight subjects at four different FFA levels. We used tritiated glucose and palmitate dilution, indirect calorimetry, glucose clamp, and forearm arteriovenous techniques together with measurements of circulating cytokines and muscle biopsies to assess insulin signaling activity.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects and study protocol (Fig. 1Go)

Eight healthy, weight-stable men (age, 24 ± 1 yr; body weight, 84 ± 9 kg; body mass index, 25 ± 3 kg/m2) with normal routine biochemistry were studied on four occasions, separated by at least 1 month. The study design has previously been used to assess the impact of FFAs on ghrelin levels (15). Informed consent was obtained from participants after oral and written information about the study. The study was conducted according to the Declaration of Helsinki II and approved by the local ethical committee.


Figure 1
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  		FIG. 1. Study protocol. See text for further details.

 
After an overnight fast, subjects were studied for 8 h from 0700 to 1500 h on four occasions with varying lipid (Intralipid 20%; Fresenius Kabi, Bad Homburg, Germany) infusions: 1) 0 µl·kg–1·min–1 (saline); 2) 3 µl·kg–1·min–1; 3) 6 µl·kg–1·min–1; and 4) 12 µl·kg–1·min–1. Three iv catheters (Venflon; Viggo AB, Helsingborg, Sweden) were inserted in a left antecubital vein, a right dorsal hand vein, and a right antecubital vein. The right hand was placed in a heated box for sampling of arterialized blood. Infusion of heparin (Heparin SAD, 70 mIE·kg–1·min–1) and somatostatin (300 µg·h–1) was initiated at t = 0 min together with replacement of GH (Norditropin; Novo Nordisk, Bagsvaerd, Denmark) (2 ng·kg–1·min–1) and glucagon (Glucagen 1 mg/ml; Novo Nordisk; 0.8 ng·kg–1·min–1). Insulin (Novo Nordisk) was infused at 0.08 mU·kg–1·min–1 for the initial 6 h of the study and continued at 0.6 mU·kg–1·min–1 during the 2-h clamp. Acipimox (Olbetam 250 mg; Pfizer, Italia S.r.l., Ascoli Piceno, Italy) was administered at t = 0 min. A primed-continuous infusion of [3-3H]-tritiated glucose (bolus, 12 µCi, 0.12 µCi·min–1) was initiated at t = 0 min and continued 8 h. To maintain euglycemia, identical glucose infusions were given from t = 60 to t = 140 min on all occasions. Blood was sampled in triplicate from 330 to 360 min (lipid) and 450–480 min (lipid + insulin). Muscle biopsies were obtained from musculi vastus lateralis at t = 480, i.e. after 2 h of hyperinsulinemia. A single basal, unstimulated biopsy was obtained from each subject in the overnight fasted state.

Tracers

Glucose flux rates were calculated at 15-min intervals during the two steady-state periods using Steele’s nonsteady-state equations (16). Endogenous glucose production (EGP) was calculated by subtracting the rate of exogenous glucose infusion from the rate of appearance of [3-3H]-tritiated glucose. Nonoxidative glucose disposal (NOGD) was calculated as glucose rate of disappearance (glucose Rd) – glucose oxidation (GOX) (17).

Systemic palmitate disposal was measured from 330 to 360 min and from 450 to 480 min during infusion of [9,10-3H] palmitate (0.3 µCi·min–1) (Department of Nuclear Medicine, Aarhus University Hospital, Aarhus, Denmark). Plasma palmitate concentration and specific activity (SA) were determined by HPLC using [2H31] palmitate as internal standard. Systemic palmitate flux (micromoles per minute) was calculated using the [9,10-3H] palmitate infusion rate (disintegrations per minute per minute) divided by the steady-state palmitate SA (disintegrations per minute per micromole). Statistical testing of regression lines through the serial measurements revealed, that the slopes were not different from zero (i.e. flat). Steady state was thus achieved (Fig. 2Go).


Figure 2
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  		FIG. 2. Palmitate SA (disintegrations per minute per nanomole) during the 4 study days. P values refer to the probability that slope = 0. As seen, steady state was achieved approximately 30 min after initiation of the [9,10-3H] palmitate infusion.

 
Intracellular signaling

Muscle biopsies were homogenized (18) and protein content in the supernatant was determined with a BCA protein assay reagent (Pierce Chemical, Rockford, IL) after centrifugation. Phosphatidylinositol 3-kinase (PI3-kinase) activity was assessed as described (18) with minor modifications. After overnight immunoprecipitation with protein A-agarose-coupled antiinsulin receptor substrate (IRS)-1 antibody (Upstate Biotechnology, Lake Placid, NY), IRS1-associated PI3-kinase activity was assessed directly on the protein A-agarose complex, resolved by thin-layer chromatography, and quantified using a phosphor imager to assess incorporation of 32P into inositol (Packard BioScience, Meriden, CT).

Aliquots of protein were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibody. Anti-Akt and antiphospho-Akt (Ser473) antibodies were obtained from New England BioLabs (Beverly, MA). AS160 phosphorylation was detected with an antiphospho-Akt substrate (New England BioLabs) as a distinct band at 160 kDa. The membranes were incubated with secondary antibody and proteins were visualized by BioWest enhanced chemiluminescent (UVP, Upland, CA) and quantified by UVP BioImaging System.

Microdialysis, indirect calorimetry, and forearm technique

At 240 min, a microdialysis catheter (CMA 60; CMA, Stockholm, Sweden) with a molecular cutoff of 20 kDa was placed in the abdominal sc adipose tissue under local anesthesia and perfused at a rate of 1 µl/min using a portable pump (CMA 106, CMA). Samples were collected from 300 min at 30-min intervals, and glucose, lactate, and glycerol concentrations were measured by an automated analyzer (CMA 600; CMA).

Indirect calorimetry was performed for 30 min at the end of the basal and insulin stimulated periods (Deltatrac; Datex Instruments, Helsinki, Finland) and rates of substrate oxidation calculated as described (17).

Catheters for measurements of forearm arteriovenous substrate balances were placed with one catheter retrogradely in a deep antecubital vein and a second catheter inserted in a heated dorsal vein. Criteria for correct positioning were oxygen saturations less than 70% and more than 91%, respectively. Hand blood flow was interrupted by a wrist cuff inflated to a pressure of 250 mm Hg 1 min before each deep venous sample.

Measurements

Serum FFAs were determined using a commercial kit (Wako Chemicals, Neuss, Germany), and plasma glucose was measured in duplicate on a glucose analyzer (Beckman Instruments, Palo Alto, CA).

An immunofluorometric assay (DELFIA, Wallac, Turku, Finland) was used to measure serum GH. Insulin and C peptide were determined by commercial kits (Dako, Glostrup, Denmark; Immunoclear, Stillwater, MN). Whole-blood glycerol, lactate, and 3-hydroxybutyrate (3-OH-butyrate) were analyzed by autofluorimetric enzymatic methods (19).

Plasma cytokines analyses were performed on the Luminex-100 system (Luminex Corp., Austin, TX) with microspheres and sheath fluid from Bio-Rad (Hercules, CA). Fluorescence data were analyzed by the Bio-plex manager software (Bio-Rad Laboratories). Plasma concentrations of IL-2, IL-4, IL-6, IL-8, IL-10, TNF-{alpha}, interferon-{gamma}, and granulocyte macrophage colony-stimulating factor were determined by the Bio-Rad 8-Plex panel (Bio-Rad Laboratories).

Statistics

Results are expressed as mean ± SEM (parametric data) or median (range) (nonparametric data). Statistical comparisons between study days were assessed by repeated-measurements ANOVA. Post hoc comparisons were performed with paired t tests or the Wilcoxon sign rank test. If the distribution of the data were skewed, data were log transformed before applying ANOVA and t tests and before calculation of correlation coefficients. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Circulating hormones and metabolites (Tables 1Go and 2Go)

Under all study conditions, circulating concentrations of insulin, glucagon, GH, cortisol, IGF-I, and IGF-II were comparable.


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  		TABLE 1. Hormones

 

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  		TABLE 2. Forearm A-V metabolites

 
FFAs. Baseline FFAs were approximately 500 µmol/liter on all study days. Antilipolysis with acipimox suppressed FFA levels (~50 µmol/liter), and by infusing intralipid, we maintained four different levels of FFAs throughout the study day: [FFAs (millimoles per liter): 0.03 ± 0.00 (LOW) vs. 0.49 ± 0.04 (NORMAL) vs. 0.92 ± 0.08 (HIGH) vs. 2.06 ± 0.33 (SUPRA), ANOVA P < 0.001]. Hyperinsulinemia decreased FFA levels on 2 study days (NORMAL, 31%, P < 0.05, and HIGH, 26%, P < 0.05), whereas the reductions observed during very low (LOW, 33%, P = not significant) and very high levels (SUPRA, 16%, P = not significant) of FFAs were non significant. Glycerol and 3-OH-butyrate concentrations increased with increasing FFAs.

Glucose. Plasma glucose levels decreased rapidly on all study days after initiation of the somatostatin infusion as described previously (5). To avoid hypoglycemia, 20% glucose was therefore infused from t = 30 to t = 60 at a rate of 15–60 ml/h. At the end of the lipid period, plasma glucose was comparable on all study days (~6.5 mmol/liter). As intended, glucose was clamped at approximately 5 mmol/liter during the hyperinsulinemic-euglycemic clamp.

Regional metabolites (microdialysis)

Interstitial fat glycerol concentration increased in parallel with increasing FFAs [glycerol (micromoles per liter): 165 ± 16 vs. 227 ± 32 vs. 299 ± 22 vs. 466 ± 46, ANOVA P < 0.001]. Hyperinsulinemia induced a similar decrease (~2.5 mmol/liter) in glucose concentration from identical levels on all study days (~5 mmol/liter) [glucose reduction (millimoles per liter): 2.11 ± 0.46 vs. 2.22 ± 0.63 vs. 2.27 ± 0.41 vs. 2.62 ± 0.51, ANOVA P = 0.86]. Neither lactate nor urea concentrations differed between study days.

Glucose metabolism and substrate oxidation (Table 3Go and Figs. 3Go and 4Go)

Lipid. Isotopically determined glucose Rd was highest at normal FFA levels (~500 µmol/liter) and significantly lower both at infraphysiological levels of FFAs and at high and supraphysiological levels. At low levels of FFAs, the low glucose Rd was primarily caused by decreased NOGD, whereas decreased GOX accounted for the low glucose Rd at high levels of FFAs (Fig. 3Go). There was no significant difference in EGP between study days.


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  		TABLE 3. Metabolic parameters

 

Figure 3
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  		FIG. 3. Glucose metabolism (A, glucose Rd; B, GOX; C, NOGD) during conditions of lipid exposure alone (left panel) and lipid plus insulin (right panel). Curves represent best fit to individual observations (Kolmogorov-Smirnov). Error bars are SEM. Statistical comparisons between study days were done with repeated-measurements ANOVA. If this test yielded a P < 0.05, post hoc comparisons were done by t test for paired samples (n = 8).

 

Figure 4
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  		FIG. 4. A, Forearm insulin-stimulated glucose A-V balances (ANOVA P < 0.05). B, EGP. *, P < 0.05 vs. lipid exposure alone. All error bars are SEM (n = 8).

 
Lipid + insulin. Hyperinsulinemia did not alter the above pattern because insulin-mediated glucose disposal remained highest at normal levels of FFAs. Again, both infraphysiological and supraphysiological levels of FFAs impaired glucose disposal (Fig. 3Go). The decreased insulin-mediated glucose disposal observed at low levels of FFAs was primarily caused by decreased NOGD, whereas supraphysiological levels of FFAs impaired both NOGD and GOX. Hepatic glucose output was significantly suppressed by hyperinsulinemia at low (79%, P < 0.05) and normal (51%, P < 0.05) levels of FFAs, whereas the suppressive effect of insulin on endogenous glucose production faded at high and supraphysiological levels of FFAs (Fig. 4Go).

Forearm metabolism

Forearm blood flow, as assessed by plethysmography, did not differ between study days, and consequently, forearm exchange of metabolites is expressed as simple arteriovenous differences in plasma concentrations of metabolites. No difference in glucose or FFA uptake was observed under conditions of lipid exposure alone: [glucoseA-V difference (LIPID) (millimoles per liter): 0.39 ± 0.09 vs. 0.30 ± 0.09 vs. 0.28 ± 0.11 vs. 0.22 ± 0.07, ANOVA P = 0.23] and [FFAA-V difference (LIPID) (millimoles per liter): 0.00 ± 0.00 vs. 0.05 ± 0.03 vs. 0.04 ± 0.03 vs. 0.00 ± 0.16, ANOVA P = 0.4] (Fig. 4Go).

Insulin-mediated forearm glucose uptake, however, was significantly affected by the various FFA levels (ANOVA P < 0.05). The pattern of results was similar to the isotopically determined glucose Rd with a peak in glucose uptake occurring at physiological levels of FFAs: [glucoseA-V difference (LIPID + INSULIN) (millimoles per liter): 1.09 ± 0.16 vs. 1.30 ± 0.20 vs. 1.15 ± 0.12 vs. 0.76 ± 0.21, ANOVA P = 0.03) (Fig. 4Go). No difference was observed in forearm FFA uptake during hyperinsulinemia: [FFAA-V difference (LIPID + INSULIN) (millimoles per liter): 0.00 ± 0.00 vs. 0.03 ± 0.01 vs. 0.10 ± 0.02 vs. 0.14 ± 0.15, ANOVA P = 0.44).

Lipid metabolism (Fig. 5Go)

The relationship between the level of FFAs and lipid oxidation followed a similar pattern regardless of insulin levels: elevation of FFAs from approximately 50 to approximately 2000 µmol/liter resulted in a rise in lipid oxidation from approximately 0.4 mg·kg–1·min–1 seemingly reaching a plateau at approximately 1 mg·kg–1·min–1. In contrast, FFA rate of disposal was linearly correlated with FFA concentration.


Figure 5
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  		FIG. 5. A, Lipid oxidation during lipid exposure (left panel) (ANOVA P < 0.05) and lipid plus insulin (right panel) (ANOVA P < 0.01). B, Palmitate turnover during lipid exposure (left panel) (ANOVA P < 0.0001) and lipid plus insulin (right panel) (ANOVA P < 0.0001). All curves represent best fit to individual observations and P values stated in the figure refer to post hoc comparisons (paired t test) between study days. Error bars are SEM (n = 8).

 
Insulin signaling

Hyperinsulinemia induced a universal approximately 1.5-fold increase (ANOVA P < 0.01) in IRS-1-associated PI3-kinase activity, protein kinase B phosphorylation, and Akt substrate 160 (AS 160) phosphorylation. However, the relationship between FFA level and enzyme activation was dose independent and PI3-kinase activity did not correlate with insulin-mediated glucose disposal (data not shown).

Cytokines

Eight hours of intralipid infusion at varying rates did not result in any differences in circulating cytokines (IL-2, IL-6, IL-10, TNF-{alpha}, or interferon-{gamma}), nor did it alter IL-4, IL-8, granulocyte macrophage colony-stimulating factor, or IL-1b (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
GH, catecholamines, and glucocorticoids have profound acute metabolic effects, notably as potent signals to increase lipolysis but also as possible mediators of insulin resistance. This study was therefore designed with the dual purpose of: 1) defining dose-response relations between FFA levels (ranging from very low to very high physiological levels) and basal and insulin-stimulated glucose and lipid metabolism, and 2) testing the hypothesis that FFA levels in the very low physiological range may cause insulin resistance. Our data demonstrate that in the basal state, glucose disposal rates peak at physiological levels (~500 µmol/liter) and decrease when FFAs levels are elevated to diabetic or acromegalic levels (~900 mmol/liter). FFA disposal is linearly related to the level of FFAs, whereas lipid oxidation reaches a maximum at FFA levels approximately 1000 µmol/liter. During insulin stimulation, glucose disposal, nonoxidative glucose disposal, and forearm muscle glucose uptake are progressively decreased at FFA levels above 3–400 µmol/liter, and in line with our preliminary hypothesis, the same parameters are suppressed at low FFA levels (~50 µmol/liter). Again, FFA disposal is linearly related to the level of FFAs, whereas lipid oxidation levels off at FFA levels approximately 1000 µmol/liter. In our setting, FFAs did not cause alterations in either proximal insulin signaling or circulating cytokines.

GH acutely increases circulating FFAs and causes insulin resistance due to decreased muscle glucose uptake, decreased oxidative and nonoxidative glucose disposal, and increased glucose production (3, 7). In the present study, we observed that elevation of FFAs above the normal basal range around 500 µmol/liter faithfully reproduced this picture, and our data therefore support the concept that GH-associated insulin resistance may be due to stimulation of lipolysis and increased FFA levels.

In the basal state, we observed a reciprocal relationship between lipid and glucose oxidation. GOX rates ranged from 1.5 to 0.8 mg/kg·min, with corresponding lipid oxidation rates from 0.5 to 1 mg/kg·min. Lipid oxidation seemed to level off at approximately 1.0 mg/kg·min, possibly because the brain depends on glucose for oxidation.

In addition, transport of FFAs into cytoplasm is partly mediated by fatty acid binding proteins (20), which is a saturable process. However, because we observed a linear relationship between the level of FFAs and palmitate/FFA turnover, a degree of passive flux of FFAs from the circulation and into tissues is also likely to occur.

Interestingly, glucose disposal rates peaked at FFA levels approximately 500 µmol/liter. This observation was very similar to our findings during hyperinsulinemia and may reflect the fact that insulin sensitivity is maximal in the physiological FFA range (~500 µmol/liter). As reported elsewhere (8, 21), short-term elevation of circulating FFAs did not alter EGP under conditions of normoinsulinemia.

Although acipimox administration effectively suppressed endogenous release of FFAs, lipids were still oxidized on the study day during which no Intralipid was infused. Other sources of lipids than FFAs must therefore be readily available for oxidation. Acipimox blocks hormone sensitive lipase (HSL)-mediated lipolysis (22), and our experimental setting thus resembles HSL-deficient knockout animal models. Recently elegant experiments demonstrated the existence of a second adipose triglyceride lipase (ATGL), which hydrolyzes triacylglycerol to diacylglycerol (DAG) (23), and it has been shown that in HSL-deficient animal models with low FFA levels, FFA reesterification is reduced (24). This provides a compensatory mechanism for FFA release but also leads to DAG accumulation in adipose tissue and muscle (25). ATGL appears to mediate basal lipolysis (26) and is up-regulated during fasting and down-regulated by insulin (27). It is thus possible that acipimox administration in this study may have induced a compensatory rise in ATGL-mediated monohydrolysis of triacylglycerol to DAG, resulting in decreased FFA reesterification, thereby providing alternative sources of FFAs for oxidation.

During the clamp we observed an impairment of insulin-mediated total glucose disposal, oxidative and nonoxidative glucose disposal, and forearm muscle glucose uptake in the 500–2.100 µmol/liter FFA range. This is in line with findings by Belfort et al. (8), who reported dose-related FFA impairment of insulin-mediated glucose disposal and proximal insulin signaling in a preclamp FFA range between 400 and 1700 µmol/liter.

Consistent with our preliminary hypothesis and observations during normoinsulinemia, at low FFA levels around 20–30 µmol/liter, we found inhibition of both insulin-mediated total glucose disposal and nonoxidative glucose disposal. To the best of our knowledge, this observation is novel. It is of interest that only nonoxidative glucose disposal was compromised, whereas oxidative glucose, if anything, was increased at low FFA levels; this distinguishes insulin resistance generated by very low FFA levels. Teleologically, the redistribution of glucose from storage to oxidation appears very beneficial in the absence of FFA fuel sources. Furthermore, insights from recent studies in HSL-deficient knockout/knockdown mice may provide a possible explanation for this apparently counterintuitive observation. HL-deficient knockout/knockdown mice display impaired insulin sensitivity during an iv glucose tolerance test in vivo and impaired insulin stimulated 2-deoxy-glucose uptake in vitro (28). There is also evidence that insulin-stimulated cardiac glucose uptake (29) is impaired in HSL-deficient rodents. The most plausible explanation for these findings is that blocking of HSL with either acipimox or insulin or by gene modification (in animals) leads to decreased FFA reesterification (26) with subsequent intracellular DAG accumulation, hypothesized to be one of the main mediators of FFA-associated insulin resistance (30). That lowering FFAs by administration of acipimox does not consistently result in improved insulin sensitivity was shown by Fulcher et al. (31), who showed that subjects treated with acipimox and intralipid/heparin (in a dose sufficient to maintain FFAs ~500 µmol/liter) had glucose uptake rates comparable with subjects treated with acipimox alone.

Our results in the 500 to more than 2000 µmol/liter FFA range confirm previous studies (6, 8, 10, 13), showing that FFAs decrease insulin-mediated glucose Rd, glucose oxidation, and nonoxidative glucose disposal. When FFAs were elevated above physiological levels, both glucose Rd and glucose uptake (assessed by A-V differences over the forearm) decreased by approximately 30%. This is consistent with results from previous studies (8, 10, 13), although slightly less than reported elsewhere (6). Our results thus provide additional evidence that FFA levels above 500 µmol/liter inhibit glucose uptake in forearm muscle dose dependently and that there is a progressive ability of FFAs to induce overt skeletal muscle insulin resistance in the full range from 500 to above 2000 µmol/liter.

Of interest, the inability of hyperinsulinemia to suppress EGP shows that hepatic insulin resistance was present in the FFA range between 700 and more than 2000 µmol/liter, confirming that an increase in plasma FFA concentration from physiological to diabetic levels impairs the insulin-mediated suppression of hepatic glucose production (32).

Contrary to the findings of some (6, 8, 10, 30), but not all (21), previous studies using lipid infusions to induce skeletal muscle insulin resistance, no significant down-regulation of IRS1-associated PI3-kinase activity, Akt/protein kinase B phosphorylation, or AS160 phosphorylation was observed in our study. However, dissociation between metabolic insulin resistance and intracellular signaling appears to be a frequent finding in experiments resembling the present. Thus, Wojtaszewski et al. (33) observed decreased insulin-stimulated IRS-1-associated PI3-kinase activity in exercised muscle, despite significantly greater insulin sensitivity. Furthermore, a recent study (12) showed time-dependent fluctuations in insulin-stimulated enzyme activity (AS160) dissociated from the actual glucose uptake observed. Finally, FFA-induced insulin resistance in impaired glucose tolerance patients and controls could not be explained by impairments of proximal insulin signal transduction after 2 h of hyperinsulinemia (21), instead of 30 min as used in most previous protocols (8, 10, 30). Taken together, these results therefore suggest that alternative mechanisms, such as distal insulin signaling defects or antecedent impairment of the proximal insulin signaling pathway, possibly contribute to insulin resistance.

Because low-grade systemic inflammation has been demonstrated in obese and insulin-resistant subjects (34, 35) and may induce insulin resistance (12), we also examined the combined effects of FFAs and hyperinsulinemia on circulating cytokines. We did not detect alterations in a wide range of cytokines.

In summary, we have shown that: 1) both very low levels and high levels of FFAs cause insulin resistance as assessed by decreased basal glucose disposal and insulin-stimulated total glucose disposal, nonoxidative glucose disposal, and forearm muscle glucose uptake; 2) during insulin exposure, EGP increases and GOX decreases dose dependently in the full range of FFA concentrations between less than 50 and more than 2000 µmol/liter; and 3) FFAs do not have measurable effects on circulating cytokine levels or the proximal insulin signaling cascade after 2 h of hyperinsulinemia. These data constitute comprehensive dose-response curves for FFAs in the full physiological range from close to zero to above 2000 µmol/liter.


   Acknowledgments
 
Microdialysis catheters were kindly supplied by Roche. The excellent technical assistance of Ms. Lone Svendsen, Ms. Elsebeth Hornemann, Ms. Elin Carstensen, and Ms. Susanne Sørensen was highly appreciated. Rasmus Strange Petersen is thanked for his help preparing the manuscript.


   Footnotes
 
This work was supported by grants from The Danish Diabetes Association, the A. P. Møller and Hustru Chastine McKinney Møllers Foundation, and The FOOD Study Group/Ministry of Food, Agriculture, and Fisheries, and Ministry of Family and Consumer Affairs.

Disclosure Summary: L.C.G., N.J., J.G., S.G., H.N., S.L., S.N., and O.S. have nothing to declare. J.S.C. has served as a consultant and received lecture fees from Novo Nordisk, Pfizer, and Ipsen. N.M. has received lecture fees from Novo Nordisk.

First Published Online March 6, 2007

Abbreviations: ATGL, Adipose triglyceride lipase; DAG, diacylglycerol; EGP, endogenous glucose production; FFA, free fatty acids; GOX, glucose oxidation; HSL, hormone-sensitive lipase; IRS, insulin receptor substrate; NOGD, nonoxidative glucose disposal; 3-OH-butyrate, 3-hydroxybutyrate; PI3-kinase, phosphatidylinositol 3-kinase; Rd, rate of disappearance; SA, specific activity.

Received December 1, 2006.

Accepted February 27, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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