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Short-Term Manipulation of Plasma Free Fatty Acids Does Not Change Skeletal Muscle Concentrations of Ceramide and Glucosylceramide in Lean and Overweight Subjects

Departments of Endocrinology and Metabolism (M.J.S., E.F., H.P.S.), Medical Biochemistry (A.J.M., J.E.G., J.M.A.), Genetic Metabolic Diseases (M.D.), and Clinical Chemistry (E.E.), Laboratory of Endocrinology, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: M.J.M. Serlie, Academic Medical Center F5-169, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: m.j.serlie{at}amc.uva.nl.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Increased plasma free fatty acid (FFA) concentrations may be in part responsible for the increased levels of ceramide in skeletal muscle of obese subjects.

Objective: We studied the effect of lowering and increasing plasma FFA levels on muscle ceramide and glucosylceramide concentrations in lean and obese subjects.

Design: Plasma FFAs were either increased or decreased for 6 h by infusing a lipid emulsion or using Acipimox, respectively. Muscle biopsies were performed before and after the intervention for measurements of ceramide and glucosylceramide.

Study Subjects: Eight lean [body mass index 21.9 (range, 19.6–24.6) kg/m²] and six overweight/obese [body mass index 34.4 (27.8–42.5) kg/m²] subjects without type 2 diabetes mellitus participated in the study.

Main Outcome Measure: Differences in muscle ceramide and glucosylceramide upon manipulation of plasma FFAs were measured.

Results: There were no differences in muscle ceramide and glucosylceramide between lean and obese subjects, respectively. Increasing or decreasing plasma FFAs for 6 h had no effect on ceramide [high FFAs: 24 (19–25) vs. 24 (22–27) pmol/mg muscle, P = 0.46; and 22 (20–28) vs. 24 (18–26) pmol/mg muscle, P = 0.89 in lean and obese, respectively; low FFAs: 26 (24–35) vs. 23 (18–27) pmol/mg muscle, P = 0.17 and 24 (15–44) vs. 24 (19–42) pmol/mg muscle, P = 0.6 in lean and obese, respectively] and glucosylceramide [high FFAs: 2.0 (1.7–4.3) vs. 3.4 (2.1–4.6) pmol/mg muscle, P = 0.17; and 3.0 (1.3–6.7) vs. 2.6 (1.2–3.9) pmol/mg muscle, P = 0.89 in lean and obese, respectively; low FFAs: 2.2 (1.0–4.4) vs. 1.7 (1.4–3.0) pmol/mg muscle, P = 0.92; and 6.6 (1.0–25.0) vs. 4.3 (1.3–7.6) pmol/mg muscle, P = 0.7 in lean and obese, respectively] concentrations in skeletal muscle.

Conclusion: Short-term manipulation of plasma FFAs has no effect on ceramide and glucosylceramide concentrations in skeletal muscle from lean and obese subjects.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
FREE FATTY ACIDS (FFAs) play a pivotal role in the induction of obesity-induced insulin resistance (1). Their effect on the insulin signaling cascade has been extensively studied. The saturated fatty acid palmitate appears to be one of the major players in the induction of reduced insulin sensitivity (2). More recently intracellular ceramide synthesis from palmitate (C16:0) and stearate (C18:0) was found to be one of the mechanisms by which palmitate negatively interferes with insulin-stimulated phosphorylation of protein kinase B (3, 4). Moreover, recent data reported an increase in ceramide concentrations in skeletal muscle of obese insulin-resistant patients (5), whereas another study found a negative correlation between whole-body insulin sensitivity and intramyocellular ceramide concentrations (6). Finally, lowering intracellular ceramide levels by overexpression of acid ceramidase in C2C12 myotubes or inhibition of serine palmitoyltransferase abolished the inhibitory effect of palmitate on insulin signaling (4, 7). An increase in intracellular ceramide may therefore account in part for the reduced insulin-mediated activation of protein kinase B in insulin resistance (8, 9). Ceramide is a precursor for gangliosides, which may also be involved in the induction of saturated fatty acid-induced insulin resistance (10). Gangliosides, like GM₃, are able to modulate insulin signaling at the level of the insulin receptor (11). Indeed GM₃ synthase knockout mice have enhanced insulin sensitivity and are protected from high-fat diet-induced insulin resistance (12). However, most studies so far were performed in cell cultures or experimental animals not necessarily representing the in vivo human situation. Therefore, we studied the effects of lowering as well as increasing plasma FFAs for 6 h with Acipimox and Intralipid, respectively, on skeletal muscle concentrations of ceramide and glucosylceramide in lean and obese subjects. We hypothesized that the earlier described decrease in peripheral glucose uptake upon increasing (13) or increase in peripheral glucose uptake upon decreasing (14) plasma FFAs for 6 h would result from increased or decreased levels of muscle ceramide or glucosylceramide, respectively.

	Subjects and Methods

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Experimental subjects

We included six overweight/obese (body mass index 27.8–42.5 kg/m²) and eight lean subjects. Participating women were not allowed to use oral contraceptives. Before inclusion, we performed an oral glucose tolerance test with 75 g glucose to exclude diabetes mellitus, defined according to the American Diabetes Association criteria. All studies were performed after an overnight fast at the Metabolic Unit of the Academic Medical Center of the University of Amsterdam. Subjects received information on carbohydrate content of commonly consumed food and consumed approximately 250 g of carbohydrates per day during 3 d before the study and were asked to refrain from vigorous exercise.

The Medical Ethical Committee of the Academic Medical Center approved the study protocol, and all participants signed a written informed consent after the nature of the study was explained.

Study protocol

Subjects were studied on two separate occasions within a time window of 4 wk. In the lean group, five subjects were studied twice and three subjects were studied once (two only-Acipimox study and one only the Intralipid study). This was done because of one withdrawal from the study and one muscle biopsy sample that was too small for a reliable analysis at t = 0 h (delta change could not be assessed in this individual). Studies with Acipimox (Altana Pharma BV, Hoofddorp, The Netherlands) and Intralipid (Fresenius Kabi Nederland BV, Den Bosch, The Netherlands) were performed in balanced assignment. Subjects were studied in the basal state after an overnight fast (t = 0 h) and after lowering or increasing plasma FFAs for 6 h (t = 6 h). At 0800 h after the subjects had fasted for 13 h, a catheter was inserted into a distal vein of each arm or a dorsal vein of the hand (during the Acipimox study, only one catheter was inserted). One catheter was used to sample arterialized blood with use of a heated hand box (60 C). The other catheter was used for the infusion of the lipid emulsion. To increase plasma FFAs, we infused Intralipid 20% without heparin at a variable rate to maintain plasma FFAs at approximately 1 mmol/liter. To achieve this, we measured plasma FFAs instantaneously every 30 min. To lower plasma FFAs, subjects received Acipimox 250 mg at t = 0 h, 250 mg at t = 2 h and 500 mg at t = 4 h. At t = 0 h and t = 6 h, blood samples were drawn for the measurements of glucose, FFAs, palmitate, glucoregulatory hormones, adiponectin, soluble TNF receptors (sTNF-Rs) I and II, ceramide and glucosylceramide. Muscle biopsies were performed at t = 0 h and t = 6 h from the musculus vastus lateralis under local anesthesia with Lidocaine 20%. Biopsies were performed using an automatic biopsy instrument (Pro-Mag I 2,5; Medical Device Technologies Inc., Gainesville, FL). The biopsies were washed with 0.9% NaCl [fortified with 10 mM Na-HEPES (pH 7.4)] to reduce blood contamination and thereafter immediately frozen in liquid nitrogen. Glucose and fat oxidation was measured at t = 0 h and t = 6 h with indirect calorimetry by using a ventilated hood system (Vmax model 2900; SensorMedics, Anaheim, CA).

Body composition

Body composition was measured with bioimpedance analysis (Maltron BF-906; Maltron International Ltd., Essex, UK).

Indirect calorimetry

Oxygen consumption (VO₂) and CO₂ production (VCO₂) were measured continuously during the final 20 min at t = 0 h and t = 6 h. The mean rates of VO₂ and VCO₂ during the final 10 min were used for calculations of the respiratory exchange ratio (RER), glucose, and fat oxidation as described below.

Analytical procedures

Insulin and cortisol were determined with an Immulite 2000 system (Diagnostic Products Corp., Los Angeles, CA). Insulin was measured by a chemiluminescent immunometric assay: intraassay variation at 47 pmol/liter 6%, 609 pmol/liter 3%; interassay variation at 91 pmol/liter 4%, 120 pmol/liter 6%; detection limit 15 pmol/liter. Cortisol was measured by a chemiluminescent immunoassay: intraassay variation at 89 nmol/liter 8%, 500 nmol/liter 7%; interassay variation at 136 nmol/liter 8%, 1092 nmol/liter 7%; detection limit 50 nmol/liter. Plasma FFAs were measured by an enzymatic method (NEFAC; Wako Chemicals, Neuss, Germany). Intraassay variation at 0.22 mmol/liter 1%, 0.93 mmol/liter 1%; interassay variation at 0.01 mmol/liter 15%, 0.48 mmol/liter 4%; detection limit 0.02 mmol/liter. Glucagon was determined with a ¹²⁵I RIA (Linco Research, Inc., St. Charles, MO). Intraassay variation at 71 ng/liter was 10%, 147 ng/liter 9%; interassay variation at 84 ng/liter was 5%, 192 ng/liter 7%; detection limit was 15 ng/liter. sTNF-RI and sTNF-RII were determined with an EASIA (Biosource Europe S.A., Nivelles, Belgium), sTNF-RI: intraassay variation at 2.31 ng/ml 7%; interassay variation at 1.76 ng/ml 6%, 26.80 ng/ml 9%; detection limit 0.05 ng/ml, sTNF-RII: intraassay variation at 5.76 ng/ml 8%; interassay variation at 3.25 ng/ml 9%, 18.70 ng/ml 7%; detection limit 0.1 ng/ml. Palmitic acid was extracted from acidified plasma (300 µl; 30 µl 4 M HCl) with ethyl acetate before its analysis by gas chromatography. The dried and evaporated sample was derivatized with 80 µl bis-(trimethylsilyl) trifluoroacetamide, containing 1% trimethylchlorosilane. The C₁₇-fatty acid was used as an internal standard. An amount equivalent to 3 µl plasma was injected into the HP5890II-gas chromatography in the split mode. Separation from the other fatty acids was achieved on a CPSil5-column (25 m x 0.25 mm, film thickness 0.25 µm). Variation coefficient 7% (n = 10).

Glucosylceramide and ceramide in plasma and muscle biopsies were measured with a HPLC method by a modification of the method described by Taketomi et al. (15). In short, muscle biopsies were weighed and homogenized in 300 µl water by sonification. To 50 µl plasma or muscle homogenates, 1 nmol of C₁₈-sphinganine was added as internal standard. Lipids were extracted according to Folch et al. (16). The lipids were hydrolyzed in borosilicate glass tubes (Schott GL14) (12 x 100 mm) with polytetrafluoretheen-lined screw caps in 0.5 ml of freshly prepared 0.1 M NaOH in methanol, using the CEM microwave solids/moisture system SAM-155 oven (CEM Corporation, Matthews, NC), equipped with a rotating Teflon tray with 36 tube holes, 60 min at 85% of maximum power. Deacylated glycosphingolipids and sphingoid bases were derivatized with 25 µl OPA reagent as described by Merrill et al. (17) with a slight modification. OPA-derivatized sphingoid bases and lysoglycosphingolipids were separated using an HPLC system (Waters Associates, Milford, MA) with a Altima BDS C₁₈ 3µ, 150 x 4.6 mm reverse-phase column and methanol-water; 88:12 (wt/wt as eluent). All samples were run in duplicate, and in every run two reference samples were included. Coefficients of variation included: interassay 4%, intraassay less than 14% (Groener, J. E., and J. M. Aerts, personal communication). Ganglioside concentrations in our muscle biopsy samples were below the detection limit.

Calculations and statistics

Glucose oxidation and fat oxidation were calculated from VO₂ and VCO₂ (18). RER was measured as VCO₂ divided by VO₂. Differences between t = 0 h and t = 6 h were calculated using the nonparametric Wilcoxon signed ranks test. Nonparametric tests were performed because we studied small groups and normal distribution of the data may not be assumed. Data are presented as medians (ranges). Differences between groups were calculated using the nonparametric Mann-Whitney U test for the basal data (t = 0 h) obtained at the first study day. Correlations were expressed as Spearman’s rank correlation coefficient (). All statistical tests were performed at a 0.05 level of significance.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subject characteristics

We included eight lean (seven males and one female) and six overweight/obese subjects (four males and two females) (Table 1).

There were no significant differences in glucoregulatory hormones between both groups, although insulin tended to be higher in the obese [lean: 66 (15–140) pmol/liter vs. obese: 134 (63–274] pmol/liter, P = 0,059]. No differences were found in fasting glucose, but there was a trend for a higher HOMA-IR in the obese subjects (Table 2). Glucose oxidation rates did not differ between groups (Table 2). After lowering plasma FFAs with Acipimox, glucose concentrations decreased significantly in both groups [lean: 5.2 (4.9–6.7) mmol/liter vs. 4.9 (4.4–5.6) mmol/liter, P = 0.04 and obese: 5.3 (4.7–7.2) mmol/liter vs. 4.6 (4.3–4.7) mmol/liter, P = 0.03]. Increasing plasma FFAs with Intralipid had no effect on plasma glucose. Glucose oxidation rates remained unchanged after Acipimox in the lean group but increased significantly in the obese group [lean: 1.99 (0.83–3.65) mg/kg fat-free mass (FFM) per minute vs. 2.68 (1.41–3.55) mg/kg FFM per minute, P = 0.18; obese: 2.04 (1.0–3.29) mg/kg FFM per minute vs. 3.26 (2.34–3.86) mg/kg FFM per minute, P = 0.03). After 6 h of Intralipid infusion, there was no change in glucose oxidation rates in both groups [lean 2.57 (1.67–3.61) mg/kg FFM per minute vs. 2.05 (1.23–3.16) mg/kg FFM per minute, P = 0.46; obese: 2.01 (0.8–3.53) mg/kg FFM per minute vs. 1.69 (0.41–2.33) mg/kg FFM per minute, P = 0,17].

Basal plasma FFAs and palmitate did not differ between groups (Table 2). Acipimox resulted in the expected decrease in plasma FFAs and palmitate in both groups (Table 3). Intralipid increased FFAs and palmitate significantly in both groups (Table 4). Fat oxidation rates did not differ between groups (Table 2). Fat oxidation rates after Acipimox decreased significantly in both groups [lean: 1.13 (0.6–1.45) mg/kg FFM per minute vs. 0.77 (0.46–1.31), P = 0.03, and obese: 1.19 (0.48–1.48) vs. 0.66 (0.51–0.73) mg/kg FFM per minute, P = 0.046]. Intralipid resulted in a significant increase in fat oxidation in the obese but not lean subjects [lean: 1.09 (0.66–1.46) mg/kg FFM per minute vs. 1.35 (1.17–1.56), P = 0.17, and obese: 1.36 (0.64–1.49) vs. 1.52 (1.38–1.95) mg/kg FFM per minute, P = 0.046].

Plasma and muscle ceramide and glucosylceramide concentrations

No differences were found in plasma or muscle ceramide and glucosylceramide concentrations between lean and obese subjects (Table 5). Acipimox resulted in a significant decrease in plasma FFAs and palmitate, but skeletal muscle ceramide and glucosylceramide did not change (Table 3). Plasma ceramide decreased significantly in the lean subjects but did not change in the obese subjects (Table 3). Intralipid increased plasma FFAs and palmitate as expected and resulted in a significant increase in plasma ceramide and glucosylceramide without changing their concentration in muscle (Table 4). Analysis of the Intralipid emulsion revealed that it contains 70 µmol/liter ceramide and 13 µmol/liter glucosylceramide.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Correlation between muscle ceramide and plasma palmitate levels. r = Spearman’s rank correlation coefficient ().

Obese subjects had higher plasma levels of sTNF-RI but not sTNF-RII (Table 2). Because TNF can stimulate ceramide generation (19, 20, 21), we correlated plasma sTNF-RI and -II levels to muscle ceramide and glucosylceramide concentrations and surprisingly found a significant negative correlation between sTNF-RI (but not sTNF-RII) with muscle ceramide (Fig. 2, A and B).

View larger version (16K): [in this window] [in a new window]   FIG. 2. A, Correlation between muscle ceramide and plasma soluble TNF-RI. B, Correlation between muscle ceramide and plasma sTNF-RII. r = Spearman’s rank correlation coefficient ().

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Extrapolating our findings to the earlier described change in peripheral insulin sensitivity upon increasing (13) or decreasing (14) plasma FFAs indicates that these changes in rate of insulin-mediated peripheral glucose uptake are not explained by changes in muscle ceramide or glucosylceramide. However, we cannot rule out a different response to high or low plasma FFA concentrations during hyperinsulinemia in ceramide and glucosylceramide synthesis. Several explanations for our findings regarding the unchanged levels of ceramide and glucosylceramide in muscle are possible. The fate of myocellular FFAs during short-term fasting differs from that in the postprandial state, i.e. oxidation vs. storage. This may have resulted in shunting away palmitate from ceramide synthesis into oxidation. However, whole-body fat oxidation did not change in the lean group and only modestly in the obese group. Therefore, we concluded that this is not the explanation for our findings. Because intracellular ceramide is involved in apoptosis (24) and macroautophagy (25), ceramide levels are tightly regulated. Therefore, increased substrate availability (palmitate) for the formation of ceramide may lead to increased channeling of palmitate into other pathways, like storage in fat or oxidation. Alternatively, if ceramide production does increase after increasing plasma palmitate levels, it may be channeled into more complex glycosphingolipids, gangliosides, or sphingomyeline.

Conversely, lowering plasma FFAs for 6 h did not lower muscle ceramide concentrations. Because de novo ceramide production from serine and palmitoyl-CoA is only one metabolic pathway for ceramide generation, other pathways may compensate, so that the levels of cellular ceramide remain constant. No significant correlation was found between plasma FFAs and muscle ceramide and glucosylceramide. Surprisingly, we did find a significant negative correlation between plasma palmitate and muscle ceramide. Because palmitate manipulation does not result in acute changes of muscle ceramide or glucosylceramide, at least during our 6-h observation, this relationship probably depends on other variables. Alternatively, in obese subjects with chronically elevated levels of palmitate, it was found that increased activity of stearoyl-CoA desaturase 1 in muscle resulted in increased desaturation of palmitate and storage into triglycerides (26). This suggests that chronically elevated palmitate leads to storage of fat instead of an increase in ceramide. Another striking finding in the present study was the higher concentrations of sTNF-RI in the obese group and the significant negative correlation between sTNF-RI and muscle ceramide. Because sTNF-RI seems to be involved in ceramide generation (20), we postulate that chronic stimulation with TNF leads to down-regulation of intracellular TNF effects regarding its effects on glycolipid metabolism. This would also explain similar muscle ceramide levels in both groups despite higher plasma sTNF-RI in the obese.

We did observe a significant increase in plasma levels of ceramide and glucosylceramide on infusion of Intralipid for 6 h. However, chemical analysis of Intralipid revealed that it contains both ceramide and glucosylceramide. Ceramide and glycosphingolipids in plasma do not exist in a free form but are associated to lipoproteins (27). They may be exchanged between lipoproteins or between cells and lipoproteins (27), but whether such a process of glycosphingolipid exchange occurs among lipoproteins, Intralipid, and myocytes is not known. Because we did not find a significant change in ceramide or glucosylceramide in skeletal muscle of our subjects, we assume that no active uptake occurred from plasma.

In summary, short-term manipulation of plasma FFAs does not affect ceramide and glucosylceramide concentrations in skeletal muscle of lean and obese subjects. This challenges a potential role for ceramide in FFA-induced insulin resistance observed in studies of short-term FFA elevation.

	Acknowledgments

 
We thank Martine van Vessem and Maarten Soeters (Department of Endocrinology and Metabolism) for their contribution to the experimental work; An Ruiter, Barbara Voermans, and Mariette Ackermans (Department of Clinical Chemistry, Laboratory of Endocrinology); and Aldi Poppema (Department of Medical Biochemistry) for analytical support.

	Footnotes

 
Disclosure Statement: M.J.S., A.J.M., J.E.G., M.D., E.E., E.F., J.M.A., and H.P.S. have nothing to declare.

First Published Online January 30, 2007

Abbreviations: FFA, Free fatty acid; FFM, fat-free mass; HOMA-IR, homeostasis model assessment of insulin resistance; RER, respiratory exchange ratio; sTNF-R, soluble TNF receptor; VCO₂, CO2 production; VO2, oxygen consumption.

Received October 26, 2006.

Accepted January 22, 2007.

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