Bioconversion of α-Linolenic Acid into n-3 Long-Chain polyunsaturated fatty acid in hepatocytes and ad hoc cell culture optimisation

This study aimed to establish optimal conditions for a cell culture system that would allow the measurement of 18:3n-3 (ALA) bioconversion into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), and to determine the overall pathway kinetics. Using rat hepatocytes (FaO) as model cells, it was established that a maximum 20:5n-3 (EPA) production from 50 mM ALA initial concentration was achieved after 3 days of incubation. Next, it was established that a gradual increase in the ALA concentration from 0 up to 125mM lead to a proportional increase in EPA, without concomitant increase in further elongated or desaturated products, such as 22:5n-3 (DPA) and 22:6n-3 (DHA) in 3 day incubations. Of interest, ALA bioconversion products were observed in the culture medium. Therefore, in vitro experiments disregarding the medium fatty acid content are underestimating the metabolism efficiency. The novel application of the fatty acid mass balance (FAMB) method on cell culture system (cells with medium) enabled quantifying the apparent enzymatic activities for the biosynthesis of n-3 LC-PUFA. The activity of the key enzymes was estimated and showed that, under these conditions, 50% (Km) of the theoretical maximal (Vmax = 3654 mmol.g21 of cell protein.hour21) Fads2 activity on ALA can be achieved with 81 mM initial ALA. Interestingly, the apparent activity of Elovl2 (20:5n-3 elongation) was the slowest amongst other biosynthesis steps. Therefore, the possible improvement of Elovl2 activity is suggested toward a more efficient DHA production from ALA. The present study proposed and described an ad hoc optimised cell culture conditions and methodology towards achieving a reliable experimental platform, using FAMB, to assist in studying the efficiency of ALA bioconversion into n-3 LC-PUFA in vitro. The FAMB proved to be a powerful and inexpensive method to generate a detailed description of the kinetics of n-3 LC-PUFA biosynthesis enzymes activities in vitro.

Measuring oral fatty acid thresholds, fat perception, fatty food liking, and papillae density in humans

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

Oral fatty acid sensitivity and dietary fat consumption

Lisa investigated the taste of fat and its influence on excess fat consumption and obesity. This research established that taste sensitivity to fat can be modulated by fat intake and may be used as an obesity prevention tool in the future.

Characterisation of lipase fatty acid selectivity using novel omega-3 pNP-acyl esters

 Lipases have applications for the industrial processing of lipids, including concentrating and/or modifying fish oil derived omega-3 fatty acids, widely used as nutritional supplement and functional food ingredients. A range of para-nitrophenol (pNP) acyl esters were synthesised as a means to rapidly screen lipases for fatty acid selectivity using spectrophotometric detection. The chosen esters were based primarily on the most abundant fatty acids present in anchovy and tuna oils. pNP derivatives of C16:1 n-7, C18:1 n-9 (OA), C18:2 n-6 (LA), C18:3 n-3 (ALA), C20:5 n-3 (EPA) and C22:6 n-3 (DHA) were synthesised. Storage stability of these pNP derivatives was shown to be at least 6 months and all pNP derivatives, including those of EPA and DHA, were shown to be stable throughout the conditions of the assay. We applied the new assay substrates for the determination of fatty acid selectivity of five widely utilised lipases. Results showed that the lipase from Candida rugosa was the most selective in terms of omega-3 specificity, preferentially hydrolysing all other medium– long chain substrates. Lipases from Rhizomucor miehei and Thermomyces lanuginosa also showed selectivity, with a significant preference for saturated fatty acids. Candida Antarctica lipase B and Aspergillus niger lipase were the least selective.

Overexpression of carnitine palmitoyltransferase I in skeletal muscle in vivo increases fatty acid oxidation and reduces triacylglycerol esterification

A key regulatory point in the control of fatty acid (FA) oxidation is thought to be transport of FAs across the mitochondrial membrane by carnitine palmitoyltransferase I (CPT I). To investigate the role of CPT I in FA metabolism, we used in vivo electrotransfer (IVE) to locally overexpress CPT I in muscle of rodents. A vector expressing the human muscle isoform of CPT I was electrotransferred into the right lateral muscles of the distal hindlimb [tibialis cranialis (TC) and extensor digitorum longus (EDL)] of rats, and a control vector expressing GFP was electrotransferred into the left muscles. Initial studies showed that CPT I protein expression peaked 7 days after IVE (+104%, P < 0.01). This was associated with an increase in maximal CPT I activity (+30%, P < 0.001) and a similar increase in palmitoyl-CoA oxidation (+24%; P < 0.001) in isolated mitochondria from the TC. Importantly, oxidation of the medium-chain FA octanoyl-CoA and CPT I sensitivity to inhibition by malonyl-CoA were not altered by CPT I overexpression. FA oxidation in isolated EDL muscle strips was increased with CPT I overexpression (+28%, P < 0.01), whereas FA incorporation into the muscle triacylglycerol (TAG) pool was reduced (−17%, P < 0.01). As a result, intramyocellular TAG content was decreased with CPT I overexpression in both the TC (−25%, P < 0.05) and the EDL (−45%, P < 0.05). These studies demonstrate that acute overexpression of CPT I in muscle leads to a repartitioning of FAs away from esterification and toward oxidation and highlight the importance of CPT I in regulating muscle FA metabolism.

Metformin counters the insulin-induced suppression of fatty acid oxidation and stimulation of triacylglycerol storage in rodent skeletal muscle

The present study examined the acute effects of metformin on fatty acid (FA) metabolism in oxidative soleus (SOL) and glycolytic epitrochlearis (EPT) rodent muscle. SOL and EPT were incubated for either 30 or 180 min in the absence or presence of 2 mM metformin and with or without insulin (10 mU/ml). Metformin did not alter basal FA metabolism but countered the effects of insulin on FA oxidation and incorporation into triacylglyerol (TAG). Specifically, metformin prevented the insulin-induced suppression of FA oxidation in SOL but did not alter FA incorporation into lipid pools. In contrast, in EPT metformin blunted the incorporation of FA into TAG when insulin was present but did not alter FA oxidation. In SOL, metformin resulted in a 50% increase in AMP-activated protein kinase α2 activity and prevented the insulin-induced increase in malonyl-CoA content. In both fiber types, basal and insulin-stimulated glucose oxidation were not significantly altered by metformin. All effects were similar regardless of whether they were measured after 30 or 180 min. Because increased muscle lipid storage and impaired FA oxidation have been associated with insulin resistance in this tissue, the ability of metformin to reverse these abnormalities in muscle FA metabolism may be a part of the mechanism by which metformin improves glucose clearance and insulin sensitivity. The present data also suggest that increased glucose clearance is not due to its enhanced subsequent oxidation. Additional studies are warranted to determine whether chronic metformin treatment has similar effects on muscle FA metabolism.

Endurance training in obese humans improves glucose tolerance and mitochondrial fatty acid oxidation and alters muscle lipid content

Muscle fatty acid (FA) metabolism is impaired in obesity and insulin resistance, reflected by reduced rates of FA oxidation and accumulation of lipids. It has been suggested that interventions that increase FA oxidation may enhance insulin action by reducing these lipid pools. Here, we examined the effect of endurance training on rates of mitochondrial FA oxidation, the activity of carnitine palmitoyltransferase I (CPT I), and the lipid content in muscle of obese individuals and related these to measures of glucose tolerance. Nine obese subjects completed 8 wk of moderate-intensity endurance training, and muscle biopsies were obtained before and after training. Training significantly improved glucose tolerance, with a reduction in the area under the curve for glucose (P< 0.05) and insulin (P = 0.01) during an oral glucose tolerance test. CPT I activity increased 250% (P = 0.001) with training and became less sensitive to inhibition by malonyl-CoA. This was associated with an increase in mitochondrial FA oxidation (+120%, P < 0.001). Training had no effect on muscle triacylglycerol content; however, there was a trend for training to reduce both the total diacylglcyerol (DAG) content (−15%, P = 0.06) and the saturated DAG-FA species (−27%, P = 0.06). Training reduced both total ceramide content (−42%, P = 0.01) and the saturated ceramide species (−32%, P < 0.05). These findings suggest that the improved capacity for mitochondrial FA uptake and oxidation leads not only to a reduction in muscle lipid content but also a to change in the saturation status of lipids, which may, at least in part, provide a mechanism for the enhanced insulin action observed with endurance training in obese individuals.

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 μM) palmitate oxidation was decreased by 95% (P < 0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P < 0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.