Acylcarnitine accumulation in skeletal muscle and plasma has been observed in numerous models of mitochondrial lipid overload and insulin resistance. Fish oil n3PUFA (omega-3 polyunsaturated fatty acids) are thought to protect against lipid-induced insulin resistance. The present study tested the hypothesis that the addition of n3PUFA to an intravenous lipid emulsion would limit muscle acylcarnitine accumulation and reduce the inhibitory effect of lipid overload on insulin action. On three occasions, six healthy young men underwent a 6-h euglycaemic–hyperinsulinaemic clamp accompanied by intravenous infusion of saline (Control), 10% Intralipid® [n6PUFA (omega-6 polyunsaturated fatty acids)] or 10% Intralipid®+10% Omegaven® (2:1; n3PUFA). The decline in insulin-stimulated whole-body glucose infusion rate, muscle PDCa (pyruvate dehydrogenase complex activation) and glycogen storage associated with n6PUFA compared with Control was prevented with n3PUFA. Muscle acetyl-CoA accumulation was greater following n6PUFA compared with Control and n3PUFA, suggesting that mitochondrial lipid overload was responsible for the lower insulin action observed. Despite these favourable metabolic effects of n3PUFA, accumulation of total muscle acylcarnitine was not attenuated when compared with n6PUFA. These findings demonstrate that n3PUFA exert beneficial effects on insulin-stimulated skeletal muscle glucose storage and oxidation independently of total acylcarnitine accumulation, which does not always reflect mitochondrial lipid overload.

CLINICAL PERSPECTIVES

  • Recent clinical studies suggest that skeletal muscle insulin resistance may arise secondary to mitochondrial lipid overload, as reflected in plasma acylcarnitine accumulation.

  • The present study provided further insight by demonstrating that the addition of fish oil to an intravenous lipid infusion in healthy male participants partially prevented lipid-induced mitochondrial overload and insulin resistance. However, these effects were not reflected in skeletal muscle total acylcarnitine content.

  • The findings confirm that acute fish oil fatty acid administration can have positive effects in insulin-resistant conditions. They also suggest that, although total acylcarnitine accumulation might not reflect mitochondrial lipid overload, a short-chain acylcarnitine might cause skeletal muscle insulin resistance. This latter target requires further investigation.

INTRODUCTION

It is generally accepted that the accumulation of lipid-derived intermediates within skeletal muscle are a major cause of insulin resistance in sedentary individuals [1,2]. However, although lipid intermediates, such as diacylglycerol, ceramides and long-chain acyl-CoAs, have the potential to impair insulin-stimulated glucose uptake, the mechanisms by which this may occur in vivo, if at all, and the characteristics (e.g. degree of saturation, chain length etc.) of any aberrant species are still not known [1,2]. Studies have suggested that lipid-induced insulin resistance may arise secondary to mitochondrial overload, whereby excess entry of fatty acids into mitochondria results in an imbalance between β-oxidation and the demands of the tricarboxylic acid cycle [25]. Under such circumstances, free carnitine, acting predominantly via CAT (carnitine acetyltransferase) and the reverse CPT2 (carnitine palmitoyltransferase 2) reactions, sequesters excess acyl groups from β-oxidation in the form of acylcarnitine, which can then be efficiently exported across the otherwise impermeable mitochondrial membrane to the cytosol [6]. Although muscle and plasma acylcarnitine accumulation has been observed in numerous models of lipid overload and insulin resistance, it is uncertain to what extent this incomplete β-oxidation can impair glucose uptake and metabolism in human skeletal muscle [5].

The intravenous infusion of lipid emulsion combined with heparin in healthy individuals during euglycaemic hyperinsulinaemia results in impaired insulin-stimulated oxidative and non-oxidative glucose disposal, with inhibition of the PDC (pyruvate dehydrogenase complex) by increased fatty acid β-oxidation and/or lipid intermediate accumulation as a primary event [7]. Interestingly, slightly varying the lipid composition of the infused emulsion in individuals with Type 2 diabetes mellitus by the addition of long-chain fish oil n3PUFA (omega-3 polyunsaturated fatty acids) has been demonstrated to partially reduce the lipid infusion-induced increase in fatty acid oxidation [8]. Furthermore, n3PUFA are thought to protect against lipid-induced skeletal muscle insulin resistance in rodents [9], and pre-incubation of primary myotubes from obese individuals with or without Type 2 diabetes mellitus with n3PUFA has been demonstrated to increase glucose oxidation compared with oleic acid [4]. Studies in human myotubes have also suggested that n3PUFA improve insulin sensitivity by diverting fatty acids away from mitochondrial β-oxidation towards cellular lipid incorporation [10]. Indeed, daily n3PUFA supplementation for 1–2 weeks has been demonstrated to reduce fat oxidation in individuals with Type 2 diabetes mellitus [11] and to improve insulin-stimulated glucose uptake [12]. However, findings from n3PUFA supplementation studies lasting 3–9 weeks are ambiguous, with most (for example, [11,13,14]) demonstrating impaired glucose uptake in individuals with Type 2 diabetes mellitus, perhaps suggesting a time-dependent effect [11]. Thus the aim of the present study was to provide further insight into the relationship between incomplete β-oxidation (skeletal muscle acylcarnitine accumulation) and insulin resistance in vivo, by testing the hypothesis that addition of n3PUFA to an intravenous lipid emulsion during 6 h of hyperinsulinaemia would limit the inhibitory effect of excessive β-oxidation on PDCa (PDC activation) and, therefore, improve insulin-stimulated glucose uptake compared with lipid emulsion alone.

MATERIALS AND METHODS

Subjects

A total of six healthy male volunteers [age, 25.7±2.3 years; body mass, 84.0±7.8 kg; BMI (body mass index), 26.8±2.4 kg/m2] gave their written informed consent to participate in the present study, which was approved by the University of Nottingham Medical School Ethics Committee in accordance with the Declaration of Helsinki.

Protocol

Volunteers reported to the laboratory following an overnight fast at 08.00 h on three randomized occasions at least 1 week apart (range, 1 to 3 weeks). On arrival, subjects were asked to rest in a supine position on a bed while cannulae were inserted into a vein in the hand for arterialized venous blood sampling, the forearm for the infusion of insulin (Actrapid; Novo Nordisk) and 20% dextrose, and the contralateral forearm for the infusion of a lipid emulsion (Intralipid® or Omegaven®; Fresenuis Kabi) or 0.9% saline, as described previously [7]. On each visit a 6-h euglycaemic–hyperinsulinaemic (50 milli-units·m−2·min−1) clamp was performed in combination with the infusion of saline (Control), 10% Intralipid® [n6PUFA (omega-6 polyunsaturated fatty acids) or 10% Intralipid®+10% Omegaven® (2:1 ratio; n3PUFA) at a rate of 100 ml/h. This insulin infusion rate was chosen as it has been demonstrated previously to completely suppress endogenous (hepatic) glucose production under insulin-resistant conditions known to affect acylcarnitine metabolism [15]. Thus the variable glucose infusion rate required to maintain euglycaemia (4.52±0.02 mmol/l) was equivalent to peripheral glucose disposal and, therefore, peripheral insulin sensitivity. The total Intralipid® infusion provided 60 g of omega-6 soya bean oil (2515 kJ), whereas the Omegaven®+Intralipid® infusion provided 20 g of highly refined omega-3 fish oil and 40 g of soya bean oil (2610 kJ). This equated into approximately 1 g of palmitic acid, 1 g of oleic acid, 10 g of linoleic acid and 1 g linolenic acid in n6PUFA being replaced with 1 g of palmitoleic acid, 1 g of arachidonic acid, 5 g of EPA (eicosapentaenoic acid), 1 g of DPA (docosapentaenoic acid) and 5 g of DHA (docosahexaenoic acid). During each lipid infusion, heparin sodium was infused at a rate of 600 units/h to elevate plasma NEFA (non-esterified fatty acid) availability.

Sample collection and analysis

Arterialized venous blood was obtained every hour for the analysis of plasma NEFA (NEFA C kit; Wako Chemicals) after the addition of tetrahydrolipstatin (30 μg/ml of plasma) on an automated analyser (ABX Pentra 400; Horiba). Plasma insulin was measured by ELISA (DRG Diagnostics). Muscle samples were also obtained from the vastus lateralis before and after each clamp using the Bergström needle biopsy technique, and immediately frozen in liquid nitrogen-cooled 2-methylbutane. One portion of the frozen muscle sample (~30 mg) was freeze-dried, separated free of visible blood, fat and connective tissue, and powdered. Acylcarnitines were then extracted using a modified version of the method described by Sun et al [16]. Briefly, powdered samples were vigorously vortex-mixed for 2 min in 500 μl of 1 M KH2PO4 buffer (pH 4.9)/propan-1-ol (1:1, v/v), and then for a further 5 min following the addition of 500 μl of acetonitrile. After centrifugation at 14000 g for 20 min at 4°C, the supernatant was removed, dried under gentle nitrogen flow and resuspended in 100 μl of propan-1-ol/1 mM acetic acid (4:1, v/v) for subsequent LC–MS analysis [17]. Acylcarnitines were screened (electrospray-positive mode) for the common carnitine moiety m/z 85 (4000 QTRAP; ABSciex) and sensitively quantified in multiple reaction monitoring mode against a dilution series of known acylcarnitine standards of various chain lengths (C2–C20). Muscle glycogen, glucose 6-phosphate and acetyl-CoA content were also determined in a portion of freeze-dried muscle powder, whereas 5–10 mg portions of frozen muscle were used to determine PDCa status, all as described previously [7]. IMCL (intramyocellular lipid) content was determined in 10-μm-thick sections cut from frozen muscle and fixed in 4% paraformaldehyde/PBS (pH 7.4). Samples were incubated at room temperature (21°C) for 1 h in 3 μM LD540/DMSO and, following 3 washes in PBS (pH 7.4), were embedded in an antifade reagent (ProLong Gold; Life Technologies) for subsequent visualization of LD540-stained lipid droplets using a TCS SP2 confocal microscope (Leica Microsystems). Briefly, 1 μm z-stacks using a 561 nm laser were captured at ×40 magnification in order to control for sample depth and background noise, and the area of the fibre covered by fluorescence was calculated using Volocity software (Volocity 6.3; PerkinElmer). LD540 is a lipophilic dye similar to BODIPY, and was manufactured by the University of Nottingham School of Chemistry according to the method described by Spandl et al. [18].

In addition, total RNA was extracted from approximately 20 mg of wet muscle tissue by the method described by Chomczynski and Sacchi [19] using TRIzol® reagent (Life Technologies). Following spectrophotometric quantification, first-strand cDNA was generated from 2 μg of RNA using the SuperScript III cDNA kit (Life Technologies) and stored at −80°C. Thereafter, the relative mRNA abundance of 24 genes from pathways involved in fatty acid metabolism and insulin signalling/carbohydrate metabolism was determined using custom-designed low-density RT–PCR (reverse transcription–PCR) array microfluidic cards (Applied Biosystems) in combination with the ABI PRISM 7900T sequence detection system and SDS 2.1 software (Applied Biosystems). The candidate genes were selected from PubMed literature searches and data obtained from our laboratory. A complete list of details for each gene assay is available in Supplementary Table S1 (at http://www.clinsci.org/cs/127/cs1270315add.htm). The threshold cycle CT was automatically given by the SDS software RQ manager, and relative mRNA abundance was calculated using the ΔΔCT method with one of the subjects’ baseline sample from their first visit as the calibrator and PPIA (encoding cyclophillin) as the endogenous control. CT values for PPIA did not change across the time points (results not shown).

Statistics

All blood and muscle data, along with acylcarnitine species grouped according to their chain length into acetyl- (C2), short-chain (C3–C5), medium-chain (C6–C10) and long-chain (C12–C20), were analysed using a two-way ANOVA (GraphPad Prism 6). When a significant main effect was detected, data were analysed further with a Student's t test using the Sidak–Bonferroni correction. Pearson's correlation coefficients with a Holm–Bonferroni stepwise correction were used to analyse associations between post-clamp acylcarnitine content and glucose disposal, glycogen accumulation and PDCa. Statistical significance was declared at P<0.05, and all of the values presented are means±S.E.M.

RESULTS

Insulin resistance

Similar steady-state (120–360 min) serum insulin concentrations of 242±12, 256±41 and 274±36 pmol/l were obtained during the hyperinsulinaemic clamps in Control, n6PUFA and n3PUFA respectively. This resulted in a complete suppression of steady-state plasma NEFA concentration during Control (0.01±0.002 mmol/l), but not during n6PUFA and n3PUFA, where lipid infusion maintained NEFA at a similar steady-state (120–360 min) concentration of 0.42±0.06 mmol/l. Despite the greater circulating NEFA concentration, IMCL content did not change following Control, n6PUFA or n3PUFA (Table 1). However, although the steady-state (240–360 min) whole-body glucose infusion rate was 28% lower in n6PUFA compared with Control (41.8±2.5 compared with 57.3±3.0 μmol·m−2·min−1; P<0.01), it was not different in n3PUFA such that it was 24% greater than n6PUFA (51.4±2.4 μmol·m−2·min−1; P<0.05 (Figure 1A). Furthermore, the greater insulin-stimulated glucose infusion rate in Control was associated with a 108±25 and 18±6% increase in PDCa (P<0.01) and muscle glycogen content (P<0.05) respectively (Figures 1B and 1C). These effects were impaired following n6PUFA, where there was no increase in muscle glycogen or PDCa, but not following n3PUFA, where muscle glycogen increased by 20±8% (P<0.05) and PDCa increased by 49±10% (P<0.05) such that it was 70±24% greater than n6PUFA (P<0.05). However, there were no differences in muscle glucose 6-phosphate content between n6PUFA and n3PUFA, which increased approximately 2-fold (P<0.05) following both (Figure 1D).

Whole-body glucose disposal (A) during 6 h of euglycaemic hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control; white squares), Intralipid® (n6PUFA; white circles) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA; black circles) infusions, and skeletal muscle PDCa status (B), glycogen content (C) and glucose 6-phophate content (D) before (white bars) and after (black bars) each infusion

Figure 1
Whole-body glucose disposal (A) during 6 h of euglycaemic hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control; white squares), Intralipid® (n6PUFA; white circles) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA; black circles) infusions, and skeletal muscle PDCa status (B), glycogen content (C) and glucose 6-phophate content (D) before (white bars) and after (black bars) each infusion

Values represent means±S.E.M. **P<0.01 compared with Control; ‡P<0.05 compared with n6PUFA; and †P<0.05 and ††P<0.01 compared with the pre-infusion value. G6P, glucose 6-phosphate; n3 lipid, n3PUFA; n6 lipid, n6PUFA.

Figure 1
Whole-body glucose disposal (A) during 6 h of euglycaemic hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control; white squares), Intralipid® (n6PUFA; white circles) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA; black circles) infusions, and skeletal muscle PDCa status (B), glycogen content (C) and glucose 6-phophate content (D) before (white bars) and after (black bars) each infusion

Values represent means±S.E.M. **P<0.01 compared with Control; ‡P<0.05 compared with n6PUFA; and †P<0.05 and ††P<0.01 compared with the pre-infusion value. G6P, glucose 6-phosphate; n3 lipid, n3PUFA; n6 lipid, n6PUFA.

Table 1
Skeletal muscle metabolite content before and after 6 h of euglycaemic hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control), Intralipid® (n6PUFA) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA) infusions

Values represent means±S.E.M., expressed as a percentage of fibre area covered for IMCL, mmol·(kg of dry muscle)−1 for acetylcarnitine, and μmol·(kg of dry muscle)−1 for acetyl-CoA and acylcarnitine (n=6). †P<0.05 and ††P<0.01 compared with the pre-infusion value; and *P<0.05 and **P<0.01 compared with the corresponding Control value.

 Control n6PUFA n3PUFA 
Content Pre-infusion Post-infusion Pre-infusion Post-infusion Pre-infusion Post-infusion 
IMCL 0.066±0.011 0.079±0.012 0.081±0.008 0.082±0.009 0.063±0.012 0.093±0.023 
Acetyl-CoA 3.9±0.9 3.9±0.5 3.8±0.9 10.1±3.3* 4.2±0.9 3.3±0.6 
Acetylcarnitine 2.6±0.6 2.0±0.7 2.5±0.8 3.7±1.2 2.2±1.0 3.2±1.4 
Short-chain acylcarnitine (C3–C526.5±6.1 15.7±4.2 25.4±4.7 40.3±15.5 27.5±4.4 29.3±3.0** 
Medium-chain acylcarnitine (C6–C105.8±1.4 1.7±0.5† 8.1±2.3 4.8±0.8 5.6±1.8 8.3±2.0** 
Long-chain acylcarnitine (C12–C2020.9±8.8 3.9±0.8† 24.6±9.6 6.9±2.2† 11.4±2.8 12.0±2.4* 
 Control n6PUFA n3PUFA 
Content Pre-infusion Post-infusion Pre-infusion Post-infusion Pre-infusion Post-infusion 
IMCL 0.066±0.011 0.079±0.012 0.081±0.008 0.082±0.009 0.063±0.012 0.093±0.023 
Acetyl-CoA 3.9±0.9 3.9±0.5 3.8±0.9 10.1±3.3* 4.2±0.9 3.3±0.6 
Acetylcarnitine 2.6±0.6 2.0±0.7 2.5±0.8 3.7±1.2 2.2±1.0 3.2±1.4 
Short-chain acylcarnitine (C3–C526.5±6.1 15.7±4.2 25.4±4.7 40.3±15.5 27.5±4.4 29.3±3.0** 
Medium-chain acylcarnitine (C6–C105.8±1.4 1.7±0.5† 8.1±2.3 4.8±0.8 5.6±1.8 8.3±2.0** 
Long-chain acylcarnitine (C12–C2020.9±8.8 3.9±0.8† 24.6±9.6 6.9±2.2† 11.4±2.8 12.0±2.4* 

Acylcarnitine metabolism

Insulin infusion suppressed total muscle acylcarnitine (sum of C3 to C20) content in Control (53.2±11.2 to 21.3±4.7 μmol/kg of dry muscle; P<0.001), but not in n6PUFA or n3PUFA (58.2±11.5 to 52.0±14.3 and 44.5±6.7 to 49.6±6.8 μmol/kg of dry muscle respectively) (Figure 2). The suppression of acylcarnitine in Control was predominantly attributable to an approximately 75% decrease in medium-chain (C6–C10; P<0.05) and long-chain (C12–C20; P<0.05) acylcarnitines (Table 1). Interestingly, this suppressive effect of insulin on long-chain acylcarnitine was also observed in n6PUFA (P<0.05; Table 1), but not in n3PUFA such that it was 3.1-fold greater than Control (P<0.05; Table 1). Similarly, medium- and short-chain acylcarnitine was 4.9- and 1.9-fold greater respectively than Control in n3PUFA (P<0.01). However, only post-clamp short-chain acylcarnitine was negatively correlated with steady-state glucose disposal across all three trials (r2=0.38, P<0.01). Of the short-chain acylcarnitines, isovaleryl- (r2=0.37, P<0.05), hydroxybutyryl/hydroxyisobutyryl- (r2=0.31, P<0.05) and propionyl-carnitine (r2=0.31, P<0.05) were negatively correlated with glucose disposal, but only isovalerylcarnitine was negatively correlated with muscle glycogen accumulation (r2=0.42, P<0.01). Short-chain acylcarnitine did not correlate with PDCa (r2=0.16). Despite muscle acetyl-CoA accumulation being 2.5-fold greater (P<0.05) following n6PUFA compared with Control and n3PUFA trials (Table 1), there were no significant changes in muscle acetylcarnitine (C2) content following Control, n6PUFA or n3PUFA (Table 1).

Skeletal muscle acylcarnitine species, and total acylcarnitine content (inset), before (Pre; horizontal hatched bars) and after 6 h of euglycaemic–hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control; white bars), Intralipid® (n6PUFA; black bars) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA; cross-hatched bars) infusions

Figure 2
Skeletal muscle acylcarnitine species, and total acylcarnitine content (inset), before (Pre; horizontal hatched bars) and after 6 h of euglycaemic–hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control; white bars), Intralipid® (n6PUFA; black bars) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA; cross-hatched bars) infusions

The Pre infusion value has been presented as the mean of all three experimental visits for clarity. Values represent means±S.E.M. †P<0.05 compared with the pre-infusion value. n3 lipid, n3PUFA; n6 lipid, n6PUFA.

Figure 2
Skeletal muscle acylcarnitine species, and total acylcarnitine content (inset), before (Pre; horizontal hatched bars) and after 6 h of euglycaemic–hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control; white bars), Intralipid® (n6PUFA; black bars) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA; cross-hatched bars) infusions

The Pre infusion value has been presented as the mean of all three experimental visits for clarity. Values represent means±S.E.M. †P<0.05 compared with the pre-infusion value. n3 lipid, n3PUFA; n6 lipid, n6PUFA.

Gene expression

Insulin infusion reduced the expression of IRS1 and IRS2 (encoding insulin receptor substrate 1 and 2 respectively), and increased the expression of PIK3R1 (encoding phosphatidylinositol 3-kinase regulatory subunit α), irrespective of lipid infusion (all time effects, P<0.01; Supplementary Table S1). On the other hand, lipid infusion reduced the expression of UCP3 (encoding uncoupling protein 3) and SLC2A4 [solute carrier family 2 (facilitated glucose transporter), member 4], and prevented the insulin-stimulated increase in SREBF1 (encoding sterol-regulatory-element-binding protein 1) expression observed in Control (all treatment or interaction effects, P<0.05; Supplementary Table S1). Interestingly, the expression of several genes responded differently to the infusion of n6PUFA and n3PUFA. NAMPT (encoding nicotinamide phosphoribosyl transferase) did not change following Control or n6PUFA, but increased in n3PUFA above baseline by 35% such that it was approximately 50% greater than Control and n6PUFA (Figure 3A). Furthermore, genes encoding peroxisome-proliferator-activated receptor α (PPARA) (Figure 3B; P<0.05) and its targets lipoprotein lipase (LPL) (Figure 3D; P<0.01) and medium-chain acyl-CoA dehydrogenase (ACADM; Figure 3E; P<0.05) were reduced following n3PUFA, but not n6PUFA, whereas PPARD (encoding peroxisome-proliferator-activated receptor δ) (Figure 3C; P<0.05) was reduced in n6PUFA, but not n3PUFA, and FASN (encoding fatty acid synthase (Figure 3E; P<0.05) was increased in n6PUFA only.

Skeletal muscle expression of genes before (white bars) and after (black bars) 6 h of euglycaemic hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control; white bars), Intralipid® (n6PUFA) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA) infusions

Figure 3
Skeletal muscle expression of genes before (white bars) and after (black bars) 6 h of euglycaemic hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control; white bars), Intralipid® (n6PUFA) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA) infusions

Values represent means±S.E.M. Only genes that were differentially expressed between n6PUFA and n3PUFA are shown. *P<0.05 compared with the corresponding Control value; ‡P<0.05 compared with the corresponding n6PUFA value; and †P<0.05 and ††P<0.01 compared with the pre-infusion value.

Figure 3
Skeletal muscle expression of genes before (white bars) and after (black bars) 6 h of euglycaemic hyperinsulinaemia (~260 pmol/l) accompanied by saline (Control; white bars), Intralipid® (n6PUFA) or Intralipid®/Omegaven® (2:1 ratio; n3PUFA) infusions

Values represent means±S.E.M. Only genes that were differentially expressed between n6PUFA and n3PUFA are shown. *P<0.05 compared with the corresponding Control value; ‡P<0.05 compared with the corresponding n6PUFA value; and †P<0.05 and ††P<0.01 compared with the pre-infusion value.

DISCUSSION

In agreement with our hypothesis, replacement of one-third of an n6PUFA lipid infusion with an equimolar fish-oil-based n3PUFA emulsion prevented much of the decline in insulin-stimulated whole-body glucose disposal and muscle PDCa, and the inhibition of muscle glycogen accumulation associated with n6PUFA. These findings provide the first evidence in humans that acute administration of n3PUFA can have beneficial effects on skeletal muscle insulin sensitivity in the face of lipid excess, albeit at a 3-fold greater dose (11 compared with 3 g of EPA+DPA+DHA) than described previously in patients with Type 2 diabetes mellitus [8]. Given the finding that some [12], but not all [11,13,14], previous studies have demonstrated improved insulin-stimulated glucose uptake with fish oil supplementation in insulin-resistant individuals and that beneficial effects on insulin sensitivity are generally observed in glucose-tolerant individuals over a short supplementation period (<2 weeks [12]) or low dose (<3 g/day [20]), further research is warranted to ascertain the optimal dose and duration of fish oil supplementation if it is to be used as a nutritional tool to improve skeletal muscle insulin sensitivity and glycaemic control in individuals with Type 2 diabetes mellitus. For example, it is unlikely that the beneficial metabolic effects of acute fish oil administration are lost during more prolonged supplementation due to progressive n3PUFA incorporation into the skeletal muscle plasma membrane, as there is a relationship between insulin sensitivity and the fatty acid composition of skeletal muscle phospholipids [21,22]. However, as acute n3PUFA administration has also been shown to divert fatty acids away from mitochondrial β-oxidation towards IMCL storage [10], one may predict that prolonged fish oil supplementation may begin to have a detrimental effect on insulin sensitivity by increasing IMCL content [1,2].

The results of the present study also provide a novel scenario of a similar amount of circulating NEFA producing a markedly different whole-body glucose disposal, allowing greater insight into the role of excessive lipid and β-oxidation in insulin resistance in humans in vivo. Thus, in line with several observational studies in insulin-resistant conditions [25,23], the greater muscle acetyl-CoA (allosteric inhibitor of the PDC) accumulation following n6PUFA suggests that excessive intramuscular β-oxidation was responsible for the lower insulin action on oxidative glucose disposal (inhibited muscle PDCa) observed compared with n3PUFA. However, the findings that muscle total and medium-chain acylcarnitine content was similar, and that long-chain acylcarnitine was actually suppressed following n6PUFA infusion compared with n3PUFA would suggest that longer-chain length acylcarnitines (C6–C20) do not accurately reflect excessive β-oxidation and insulin resistance. This is in agreement with the finding of Soeters et al. [15] that muscle long-chain acylcarnitines did not reflect fasting-induced insulin resistance in humans. On the other hand, muscle short-chain acylcarnitine levels tended to be higher after n6PUFA infusion and negatively correlated with whole-body glucose disposal, with C5 (isovaleryl), C4OH (hydroxybutyryl/hydroxyisobutyryl) and C3 (propionyl) carnitine having the strongest relationship. However, it is important to note that these acylcarnitines are derived from BCAA (branched-chain amino acid) catabolism and not fatty acid β-oxidation, providing further evidence against incomplete β-oxidation (i.e. acylcarnitine accumulation as opposed to excessive acetyl-CoA accumulation) causing or reflecting insulin resistance. This is consistent with other studies demonstrating a strong association of hydroxybutyrylcarnitine [24,25] and other short-chain acylcarnitines of BCAA catabolism [26,27] with insulin-resistant states and the development of Type 2 diabetes mellitus [28]. Indeed, the ability of insulin to suppress BCAA catabolism is impaired in insulin resistance [29], and fatty acids have been shown to activate branch-chain keto-acid dehydrogenase (the rate limiting step in BCAA oxidation [30]). It is also important to note that, unlike short-chain acyl-CoA products of mitochondrial BCAA catabolism, short-chain acylcarnitines do not inhibit PDC activity in vitro [31]. In support of the latter, short-chain acylcarnitine products of mitochondrial BCAA catabolism did not negatively correlate with PDCa in the present study, suggesting that acetyl-CoA is more important for inducing insulin resistance at the level of glucose oxidation. Of course, we also observed a beneficial effect of n3PUFA infusion on non-oxidative glucose disposal in that there was a complete prevention of the inhibition of net glycogen synthesis observed in n6PUFA. Thus whether short-chain acylcarnitines that are exported from mitochondria directly inhibited glucose uptake or storage and caused insulin resistance in the present study clearly requires further investigation, particularly as isovalerylcarnitine had a negative correlation with glycogen accumulation and acylcarnitines have been demonstrated previously to stimulate key cytosolic pathways implicated in lipid-induced skeletal muscle insulin resistance [32].

The differences in skeletal muscle fatty acid metabolism between n6PUFA and n3PUFA in the present study are in agreement with previous cell and animal studies [4,9,10], and are supported by the finding that n3PUFA suppressed the expression of PPARA, which is a key transcription factor controlling the expression of genes involved in fatty acid oxidation, and MCAD (encoding medium-chain acyl-CoA dehydrogenase), which is a rate-limiting step in β-oxidation downstream of medium- and long-chain acylcarnitine accumulation. Indeed, the PPAR transcription factors are known to have different affinities for saturated, mono-unsaturated and polyunsaturated fatty acids [33], and this differential expression of genes between n6PUFA and n3PUFA infusion was also observed for other genes involved in fatty acid metabolism including PPARD, NAMPT, LPL and FASN. In addition, although not significantly different, IMCL content increased by approximately 50% (range, −13.4 to 264.4%) in n3PUFA infusion in the present study, which would fit with the suggestion of fatty acids being diverted from oxidation toward cellular lipid incorporation [10], particularly as NAMPT has been shown to be responsive to changes in cellular triacylglycerol (triglyceride) and phospholipid metabolism [34], the gene expression of which increased in every volunteer during the n3PUFA visit. However, genes involved in insulin signalling and glucose metabolism that changed in the present study such as IRS1, IRS2, PIK3R1, SREBF1 and SLC2A4 were not differentially expressed between n6PUFA and n3PUFA, which is in line with cell-based studies [10], possibly because the proteins encoded by these genes are regulated by phosphorylation or translocation.

In conclusion, replacement of predominantly n6PUFA linoleic acid with n3PUFA fish oil fatty acids in an intravenous lipid emulsion infusion during 6 h of hyperinsulinaemia improves insulin-stimulated oxidative and non-oxidative glucose disposal compared with lipid emulsion alone. This was probably due to a reduction in the inhibitory effect of excessive β-oxidation on PDCa, and possibly the inhibitory effect of lipid on glucose transport and storage by an unidentified mechanism. The model used in the present study provides further human in vivo evidence of excess entry of fatty acids into the mitochondria causing insulin resistance, but is in contrast with that speculated in the literature that skeletal muscle total acylcarnitine accumulation from incomplete β-oxidation may be a causative factor. Nevertheless, the effect of short-chain acylcarnitines from aberrant amino acid metabolism on insulin action in skeletal muscle requires further investigation.

We thank Dr Dan Speed (University of Nottingham School of Chemistry) for synthesizing the LD540 dye.

Abbreviations

     
  • ACADM

    medium-chain acyl-CoA dehydrogenase

  •  
  • BCAA

    branched-chain amino acid

  •  
  • DHA

    docosahexaenoic acid

  •  
  • DPA

    docosapentaenoic acid

  •  
  • EPA

    eicosapentaenoic acid

  •  
  • FASN

    fatty acid synthase

  •  
  • IMCL

    intramyocellular lipid

  •  
  • IRS1

    insulin receptor substrate 1

  •  
  • IRS2

    insulin receptor substrate 2

  •  
  • LPL

    lipoprotein lipase

  •  
  • n3PUFA

    omega-3 polyunsaturated fatty acid(s)

  •  
  • n6PUFA

    omega-6 polyunsaturated fatty acid(s)

  •  
  • NAMPT

    nicotinamide phosphoribosyl transferase

  •  
  • NEFA

    non-esterified fatty acid

  •  
  • PDC

    pyruvate dehydrogenase complex

  •  
  • PDCa

    PDC activation

  •  
  • PIK3R1

    phosphatidylinositol 3-kinase regulatory subunit α

  •  
  • PPARA

    peroxisome-proliferator-activated receptor α

  •  
  • PPARD

    peroxisome-proliferator-activated receptor δ

  •  
  • PPIA

    cyclophillin

  •  
  • SLC2A4

    solute carrier family 2 (facilitated glucose transporter), member 4

  •  
  • SREBF1

    sterol-regulatory-element-binding protein 1

AUTHOR CONTRIBUTION

Francis Stephens, Peter Mansell and Kostas Tsintzas designed the research. Francis Stephens drafted the paper and had primary responsibility for final content. All of the authors contributed to the acquisition, analysis and interpretation of the data, revised the paper for important intellectual content, and approved the final version.

FUNDING

This work was supported by The Royal Society [grant number RG100575].

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Supplementary data