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.

Seasonal variations of lipid content and composition in perna viridis

The total lipid content, composition of main lipid classes, composition of sterols and composition of fatty acids in the main glycerolipids of Perna viridis were analyzed through four seasons using TLC-FID and GLC. Mussel samples were collected during different seasons between 2003 and 2004 from Shengsi Island, Zhejiang Province, China and stored frozen prior to freeze-drying and lipid extraction. Ten grams of dried mussel powder of each season were analyzed. Total lipid content ranged from 14.5 g/100 g in spring month to 7.8 g/100 g dried mussel powder in autumn month. The predominant lipid in spring month was triacylglycerol (TAG), however, in the other three seasons the phospholipids (PL) was the main lipid class. The most abundant fatty acid in TAG, PL and phosphatidylcholine (PC) was 16:0, with the summer samples having the highest proportion (24-30% of total fatty acid) and winter the lowest (14-22%). In phosphatidylethanolamine (PE), the spring samples had the highest proportions of 16:0. The predominant polyunsaturated fatty acids (PUFA) were 22:6n-3 and 20:5n-3 in TAG, PL, PE and PC (25-40%). The proportions of 22:6n-3 and 20:5n-3 were higher in spring than in other seasons in PL and PE. There were nine sterols identified, with cholesterol being the predominant sterol, and other main ones were desmostersol/brassicasterol and 24-methylenecholesterol. Proportions of other fatty acids in different lipid fractions and the sterol compositions as well also varied seasonally. There were subject to the seasonal variations. Differences in lipid content and composition, fatty acid composition in different lipid fractions may be caused by multiple factors such as lifecycle, sex, variation of plankton in different seasons and temperature, which could influence physiological activities and metabolism.

Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites

Consumption of a Western diet rich in saturated fats is associated with obesity and insulin resistance. In some insulin-resistant phenotypes this is associated with accumulation of skeletal muscle fatty acids. We examined the effects of diets high in saturated fatty acids (Sat) or n-6 polyunsaturated fatty acids (PUFA) on skeletal muscle fatty acid metabolite accumulation and whole-body insulin sensitivity. Male Sprague-Dawley rats were fed a chow diet (16% calories from fat, Con) or a diet high (53%) in Sat or PUFA for 8 wk. Insulin sensitivity was assessed by fasting plasma glucose and insulin and glucose tolerance via an oral glucose tolerance test. Muscle ceramide and diacylglycerol (DAG) levels and triacylglycerol (TAG) fatty acids were also measured. Both high-fat diets increased plasma free fatty acid levels by 30%. Compared with Con, Sat-fed rats were insulin resistant, whereas PUFA-treated rats showed improved insulin sensitivity. Sat caused a 125% increase in muscle DAG and a small increase in TAG. Although PUFA also resulted in a small increase in DAG, the excess fatty acids were primarily directed toward TAG storage (105% above Con). Ceramide content was unaffected by either high-fat diet. To examine the effects of fatty acids on cellular lipid storage and glucose uptake in vitro, rat L6 myotubes were incubated for 5 h with saturated and polyunsaturated fatty acids. After treatment of L6 myotubes with palmitate (C16:0), the ceramide and DAG content were increased by two- and fivefold, respectively, concomitant with reduced insulin-stimulated glucose uptake. In contrast, treatment of these cells with linoleate (C18:2) did not alter DAG, ceramide levels, and glucose uptake compared with controls (no added fatty acids). Both 16:0 and 18:2 treatments increased myotube TAG levels (C18:2 vs. C16:0, P < 0.05). These results indicate that increasing dietary Sat induces insulin resistance with concomitant increases in muscle DAG. Diets rich in n-6 PUFA appear to prevent insulin resistance by directing fat into TAG, rather than other lipid metabolites.

A short-term n-3 DPA supplementation study in humans

Purpose Despite the detailed knowledge of the absorption and incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into plasma lipids and red blood cells (RBC) in humans, very little is known about docosapentaenoic acid (DPA, 22:5 n-3). The aim of this study was to investigate the uptake and incorporation of pure DPA and EPA into human plasma and RBC lipids.

Methods Ten female participants received 8 g of pure DPA or pure EPA in randomized crossover double-blinded manner over a 7-day period. The placebo treatment was olive oil. Blood samples were collected at days zero, four and seven, following which the plasma and RBC were separated and used for the analysis of fatty acids.

Results Supplementation with DPA significantly increased the proportions of DPA in the plasma phospholipids (PL) (by twofold) and triacylglycerol (TAG) fractions (by 2.3-fold, day 4). DPA supplementation also significantly increased the proportions of EPA in TAG (by 3.1-fold, day 4) and cholesterol ester (CE) fractions (by 2.0-fold, day 7) and of DHA in TAG fraction (by 3.1-fold, day 4). DPA proportions in RBC PL did not change following supplementation. Supplementation with EPA significantly increased the proportion of EPA in the plasma CE and PL fractions, (both by 2.7-fold, day 4 and day 7) and in the RBC PL (by 1.9-fold, day 4 and day 7). EPA supplementation did not alter the proportions of DPA or DHA in any lipid fraction. These results showed that within day 4 of supplementation, DPA and EPA demonstrated different and specific incorporation patterns.

Conclusion The results of this short-term study suggest that DPA may act as a reservoir of the major long-chain n-3 fatty acids (LC n-3 PUFA) in humans.

Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA

Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years. By expressing a transgenic pathway for enhancing the synthesis of the ω3 LC-PUFA docosahexaenoic acid (DHA) from endogenous α-linolenic acid (ALA), we obtained the production of fish oil-like proportions of DHA in Arabidopsis seed oil. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages. The analysis identified the appearance of several abundant DHA-containing phosphatidylcholine (PC), DAG and TAG molecular species in mature seeds. The relative abundances of PL, DAG, and TAG species showed a preferred combination of LC-PUFA with ALA in the transgenic seeds, where LC-PUFA were esterified in positions usually occupied by 20:1ω9. Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS. Studying the lipidome in transgenic seeds provided insights into where DHA accumulated and combined with other fatty acids of neutral and phospholipids from the developing and mature seeds. © 2014 Zhou, Callahan, Shrestha, Liu, Petrie and Singh.

Tristearin bilayers: structure of the aqueous interface and stability in the presence of surfactants

We report results of atomistic molecular dynamics simulations of an industrially-relevant, exemplar triacylglycerol (TAG), namely tristearin (TS), under aqueous conditions, at different temperatures and in the presence of an anionic surfactant, sodium dodecylbenzene sulphonate (SDBS). We predict the TS bilayers to be stable and in a gel phase at temperatures of 350 K and below. At 370 K the lipid bilayer was able to melt, but does not feature a stable liquid-crystalline phase bilayer at this elevated temperature. We also predict the structural characteristics of TS bilayers in the presence of SDBS molecules under aqueous conditions, where surfactant molecules are found to spontaneously insert into the TS bilayers. We model TS bilayers containing different amounts of SDBS, with the presence of SDBS imparting only a moderate effect on the structure of the system. Our study represents the first step in applying atomistic molecular dynamics simulations to the investigation of TAG-aqueous interfaces. Our results suggest that the CHARMM36 force-field appears suitable for the simulation of such systems, although the phase behaviour of the system may be shifted to lower temperatures than is the case for the actual system. Our findings provide a foundation for further simulation studies of the TS-aqueous interface.

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.

Reduced triacylglycerol mobilization during seed germination and early seedling growth in Arabidopsis containing nutritionally important polyunsaturated fatty acids

There are now several examples of plant species engineered to synthesize and accumulate nutritionally important polyunsaturated fatty acids in their seed triacylglycerols (TAG). The utilization of TAG in germinating seeds of such transgenic plants was unknown. In this study, we examined the TAG utilization efficiency during seed germination in transgenic Arabidopsis seeds containing several examples of these fatty acids. Seed TAG species with native fatty acids had higher utilization rate than the TAG species containing transgenically produced polyunsaturated fatty acids. Conversely, quantification of the fatty acid components remaining in the total TAG after early stages of seed germination revealed that the undigested TAGs tended to contain elevated levels of the engineered polyunsaturated fatty acids (PUFA). LC-MS analysis further revealed asymmetrical mobilization rates for the individual TAG species. TAGs which contained multiple PUFA fatty acids were mobilized slower than the species containing single PUFA. The mobilized engineered fatty acids were used in de novo membrane lipid synthesis during seedling development.

The occurrence of trans-18:1 isomers in plasma lipids classes in humans

Objective: The aim of this study was to examine the distribution of trans fatty acids (TFA) in plasma lipid classes and the relationship with dietary intake of TFA.

Design: After a 2 week baseline (habitual) diet, all subjects consumed a moderate fat (MF) diet for 3 weeks with the fat being derived mainly from margarine and the rest from lean beef, and then a very low fat (VLF) diet for 3 weeks with the TFA being derived only from the lean beef. Blood samples were collected 2 days prior to the end and also on the last day of each dietary period.

Setting: Deakin Institute of Human Nutrition, Deakin University, Geelong, Australia.

Subjects: Ten free-living mildly hypercholesterolaemic subjects aged 22-66 were recruited in Geelong.

Outcome measures: TFA intake was calculated from analyses of Australian margarines, butter, lean meat and animal fat. The TFA in plasma lipid fractions were separated by AgNO3 thin-layer chromatography and quantitated by capillary gas-liquid chromatography using internal standards.

Results: The phospholipid (PL) fraction contained more than 60% of the trans-18:1 isomers in the plasma lipids in all subjects. On the baseline diet, the predominant positional isomer of trans-18:1 in PL was Delta11, whereas in the other lipid classes it was the Delta9 isomer. The concentration of the Delta9 isomer increased on the MF diet, particularly in the PL fraction, while the concentration of the Delta11 isomer decreased in all fractions. On the VLF diet, the total TFA level decreased by approximately 50%, mainly due to decreases in the TFA isomers in the PL and TG fractions. Changes in plasma total and PL TFA, PL Delta9, Delta10 and Delta11 were strongly correlated with dietary TFA intake (P<0.0001). There were also significant association between dietary TFA intake and PL Delta12 (P=0.003), triacylglycerol Delta9 (P=0.009), Delta11 (P=0.0005), total triacylglycerol (P=0.023) and free fatty acid TFA (P=0.042).

Conclusions: The results suggest that the measurement of trans-18:1 in plasma PL and TAG, and plasma total TFA could be used to estimate the intake of TFA.

Enhancing RFID tag resistance against cloning attack

In its current form, RFID system are susceptible to a range of malevolent attacks. With the rich business intelligence that RFID infrastructure could possibly carry, security is of paramount importance. In this paper, we formalise various threat models due tag cloning on the RFID system. We also present a simple but efficient and cost effect technique that strengthens the resistance of RFID tags to cloning attacks. Our techniques can even strengthen tags against cloning in environments with untrusted reading devices.

Effect of a stearic acid-rich, structured triacylglycerol on plasma lipid concentrations

Background: Structured lipids are being incorporated into foods to reduce their energy value. One such lipid is rich in stearic acid.
Objective: The objective of this study was to compare the effects on plasma lipids of a stearic acid–rich triacylglycerol and a fat rich in palmitic acid in hypercholesterolemic subjects.
Design: Fifteen subjects with an average plasma cholesterol concentration of 6.13 ± 0.80 mmol/L initially ate a low-fat diet for 2 wk (run-in period), followed in random order and blinded fashion by 2 high-fat diets (for 5 wk each) containing foods derived from margarines rich either in palmitic acid or in the structured, stearic acid–rich triacylglycerol.
Results: Plasma cholesterol concentrations with the low-fat, the stearic acid–rich, and the palmitic acid–rich diets were not significantly different (5.35 ± 0.83, 5.41 ± 0.78, and 5.52 ± 0.68 mmol/L, respectively) but were significantly lower (P < 0.001) than those measured during the habitual diet period (ie, 2 wk before the study began). Neither HDL cholesterol nor plasma triacylglycerol differed significantly among the 3 study diets.
Conclusion: A similar increase in the intake of stearic and palmitic acids (differing by <5% of total energy) to ensure a high fat intake resulted in plasma total and LDL-cholesterol concentrations that did not differ significantly from concentrations measured during a period of low-fat intake.

Production of Omega-3 Triacylglycerol concentrates using a new food grade immobilized Candida antarctica Lipase B

Triacylglycerol concentrates of eicosapentaenoic and docosahexaenoic omega-3 fatty acids were synthesized either via transesterification or esterification of glycerol with the corresponding ethyl ester or free fatty acid concentrates, respectively. A newly developed food grade immobilized Candida antarctica lipase Β system using an Amberlite FPX-66 hydrophobic matrix, was compared with a commercially available non-food grade commercial system, for their ability to catalyze these reactions. For either system, the transesterification required higher temperature (90◦C) than esterification (70°C) to achieve maximum triacylglycerol yields. The newly developed immobilized system efficiently catalyzes the esterification of free fatty acids with glycerol and differs from the existing commercial system in that it is food grade and has a more uniform and larger particle distribution. The new system significantly improves flow in a packed bed reactor, enabling multiple reuse of the catalyst for up to 80 repeats.

A secure tag ownership transfer scheme in a closed loop RFID system

In this paper we propose a novel secure tag ownership transfer scheme for closed loop RFID systems. An important property of our method is that the ownership transfer is guaranteed to be atomic and the scheme is protected against desynchronisation leading to permanent DoS. Further, it is suited to the computational constraints of EPC Class-1 Gen-2 passive RFID tags as they only use the CRC and PRNG functions that passive RFID tags are capable of. We provide a detailed security analysis to show that our scheme satisfies the required security properties of tag anonymity, tag location privacy, forward secrecy, forward untraceability while being resistant to replay, desynchronisation and server impersonation attacks. Performance comparisons show that our scheme is practical and can be implemented on passive low-cost RFID tags.

StenoCipher to provide data confidentiality and tampered data recovery for RFID tag

Radio Frequency Identification (RFID) is an emerging wireless object identification technology with many potential applications such as supply chain management, personnel tracking and healthcare. However, security vulnerabilities of the RFID system have been a serious concern for its wide adoption in many applications. Although much work has been done to provide privacy and anonymity, little focus has been given to ensure RFID data confidentiality, integrity and to address the tampered data recovery problem. To this end, we propose a lightweight stenographic-based approach to ensure RFID data confidentiality and integrity as well as the recovery of tampered RFID data.