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.

Cytokine regulation of skeletal muscle fatty acid metabolism: effect of interleukin-6 and tumor necrosis factor-α

IL-6 and TNF-α have been associated with insulin resistance and type 2 diabetes. Furthermore, abnormalities in muscle fatty acid (FA) metabolism are strongly associated with the development of insulin resistance. However, few studies have directly examined the effects of either IL-6 or TNF-α on skeletal muscle FA metabolism. Here, we used a pulse-chase technique to determine the effect of IL-6 (50-5,000 pg/ml) and TNF-α (50-5,000 pg/ml) on FA metabolism in isolated rat soleus muscle. IL-6 (5,000 pg/ml) increased exogenous and endogenous FA oxidation by ≃50% (P < 0.05) but had no effect on FA uptake or incorporation of FA into endogenous lipid pools. In contrast, TNF-α had no effect on FA oxidation but increased FA incorporation into diacylglycerol (DAG) by 45% (P < 0.05). When both IL-6 (5,000 pg/ml) and insulin (10 mU/ml) were present, IL-6 attenuated insulin's suppressive effect on FA oxidation, increasing exogenous FA oxidation (+37%, P < 0.05). Furthermore, in the presence of insulin, IL-6 reduced the esterification of FA to triacylglycerol by 22% (P < 0.05). When added in combination with IL-6 or leptin (10 μg/ml), the TNF-α-induced increase in DAG synthesis was inhibited. In conclusion, the results demonstrate that IL-6 plays an important role in regulating fat metabolism in muscle, increasing rates of FA oxidation, and attenuating insulin's lipogenic effects. In contrast, TNF-α had no effect on FA oxidation but increased FA incorporation into DAG, which may be involved in the development of TNF-α-induced insulin resistance in skeletal muscle.

Disassociation of muscle triglyceride content and insulin sensitivity after exercise training in patients with Type 2 diabetes

Aim/hypothesis. We determined the effect of exercise training on insulin sensitivity and muscle lipids (triglyceride [TGm] and long-chain fatty acyl CoA [LCACoA] concentration) in patients with Type 2 diabetes. Methods. Seven patients with Type 2 diabetes and six healthy control subjects who were matched for age, BMI, % body fat and VO2peak participated in a 3 days per week training program for 8 weeks. Insulin sensitivity was determined pre- and post-training during a 120 min euglycaemic- hyperinsulinaemic clamp and muscle biopsies were obtained before and after each clamp. Oxidative enzyme activities [citrate synthase (CS), β-hydroxy-acyl- CoA (β-HAD)] and TGm were determined from basal muscle samples pre- and post training, while total LCACoA content was measured in samples obtained before and after insulin-stimulation, pre- and post training. Results. The training-induced increase in VO2peak (∼20%, p<0.01) was similar in both groups. Compared with control subjects, insulin sensitivity was lower in the diabetic patients before and after training (∼60%; p<0.05), but was increased to the same extent in both groups with training (∼30%; p<0.01). TGm was increased in patients with Type 2 diabetes (170%; p<0.05) before, but was normalized to levels observed in control subjects after training. Basal LCACoA content was similar between groups and was unaltered by training. Insulin-stimulation had no detectable effect on LCACoA content. CS and β-HAD activity were increased to the same extent in both groups in response to training (p<0.001). Conclusion/interpretation. We conclude that the enhanced insulin sensitivity observed after short-term exercise training was associated with a marked decrease in TGm content in patients with Type 2 diabetes. However, despite the normalization of TGm to levels observed in healthy individuals, insulin resistance was not completely reversed in the diabetic patients.

Overexpression of sphingosine kinase 1 in liver reduces triglyceride content in mice fed a low but not high-fat diet

 Hepatic insulin resistance is a major risk factor for the development of type 2 diabetes and is associated with the accumulation of lipids, including diacylglycerol (DAG), triacylglycerols (TAG) and ceramide. There is evidence that enzymes involved in ceramide or sphingolipid metabolism may have a role in regulating concentrations of glycerolipids such as DAG and TAG. Here we have investigated the role of sphingosine kinase (SphK) in regulating hepatic lipid levels. We show that mice on a high-fat high-sucrose diet (HFHS) displayed glucose intolerance, elevated liver TAG and DAG, and a reduction in total hepatic SphK activity. Reduced SphK activity correlated with downregulation of SphK1, but not SphK2 expression, and was not associated with altered ceramide levels. The role of SphK1 was further investigated by overexpressing this isoform in the liver of mice in vivo. On a low-fat diet (LFD) mice overexpressing liver SphK1, displayed reduced hepatic TAG synthesis and total TAG levels, but with no change to DAG or ceramide. These mice also exhibited no change in gluconeogenesis, glycogenolysis or glucose tolerance. Similarly, overexpression of SphK1 had no effect on the pattern of endogenous glucose production determined during a glucose tolerance test. Under HFHS conditions, normalization of liver SphK activity to levels observed in LFD controls did not alter hepatic TAG concentrations. Furthermore, DAG, ceramide and glucose tolerance were also unaffected. In conclusion, our data suggest that SphK1 plays an important role in regulating TAG metabolism under LFD conditions.

Changes in the nutritional composition of captive early-mid stage Panulirus ornatus phyllosoma over ecdysis and larval development

Elucidation of the key nutritional requirements for complete larval development of the tropical spiny rock lobster, Panulirus ornatus, presents a major challenge for the development of robust commercial aquaculture for this crustacean. As a foundation study in this area, the chemical composition of early-mid stage P. ornatus phyllosoma (Stages I-VI) receiving a novel formulated diet was analysed immediately prior and post-ecdysis to provide insight into the crude nutritional trends during the larval development cycle. From the onset of moulting, cyclical patterns were evident in the proximate composition of phyllosoma, resulting in substantial restructuring between the pre- and post-moult stages of the moult cycle. Proportions of protein, lipid and ash were high at the premoult stage, reflecting growth and nutrient accumulation over the intermoult period, and reduced at the post-moult stage, reflecting the large uptake of water to facilitate subsequent growth. Polar lipid was the dominant lipid class, accounting for >. 90% of the total lipid content. Conversely, triacylglycerol concentrations were low (<. 5%), despite being the principal lipid class available in the formulated diet. Likewise, despite receiving high concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) (9.2 and 7.6% of the dietary lipid source, respectively), levels of these fatty acids were comparatively low in phyllosoma (3.4 and 4.7%, respectively). In contrast, there is selective deposition of these fatty acids in wild caught phyllosoma. This finding suggests a poor assimilation of triacylglycerols by captive larvae and highlights the importance of future investigations into alternative sources of EPA and DHA. Ultimately, this study provides insight into the nutritional requirements of phyllosoma, providing valuable knowledge on diet formulation for commercially viable hatchery production of spiny rock lobsters. © 2014 Elsevier B.V.

Is fat the sixth taste primary? Evidence and implications

Taste is the chemical sense responsible for the detection of non-volatile chemicals in potential foods. For fat to be considered as one of the taste primaries in humans, certain criteria must be met including: class of affective stimuli; receptors specific for the class of stimuli on taste bud cells (TBC); afferent fibers from TBC to taste processing regions of the brain; perception independent of other taste qualities; and downstream physiological effects. The breakdown products of the macronutrients carbohydrates (sugars) and protein (amino acids) are responsible for activation of sweet and umami tastes respectively. Following the same logic the breakdown products of fat being fatty acids are the likely class of stimuli for fat taste. Indeed, psychophysical studies have confirmed fatty acids of varying chain length and saturation are orally detectable by humans. The most likely fatty acid receptor candidates located on TBC are CD36, G protein-coupled receptor 120. Once the receptors are activated by fatty acids a series of transduction events occurs causing the release of neurotransmitters towards afferent fibers signalling the brain. Whether fatty acids elicit any direct perception independent of other taste qualities is still open to debate with only poorly defined perceptions for fatty acids reported. Others suggest that the fatty acid taste component is at detection threshold only and any perceptions are associated with either aroma or chemesthesis. It has also been established that oral exposure to fat via sham feeding stimulates increases blood triacylglycerol concentrations in humans. Therefore, overall, with the exception of an independent perception, there is consistent emerging evidence that fat is the sixth taste primary. The implications of fatty acid taste go further into health and obesity research with the gustatory detection of fats and their contributions to energy and fat intake receiving increasing attention. There appears to be a coordinated bodily response to fatty acids throughout the alimentary canal; those who are insensitive orally are also insensitive in the gastrointestinal tract and overconsume fatty food and energy. The likely mechanism linking fatty acid taste insensitivity with overweight and obesity is development of satiety after consumption of fatty foods.

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.

PLIN5 deletion remodels intracellular lipid composition and causes insulin resistance in muscle

Defective control of lipid metabolism leading to lipotoxicity causes insulin resistance in skeletal muscle, a major factor leading to diabetes. Here, we demonstrate that perilipin (PLIN) 5 is required to couple intramyocellular triacylglycerol lipolysis with the metabolic demand for fatty acids. PLIN5 ablation depleted triacylglycerol stores but increased sphingolipids including ceramide, hydroxylceramides and sphingomyelin. We generated perilipin 5 (Plin5)-/- mice to determine the functional significance of PLIN5 in metabolic control and insulin action. Loss of PLIN5 had no effect on body weight, feeding or adiposity but increased whole-body carbohydrate oxidation. Plin5-/- mice developed skeletal muscle insulin resistance, which was associated with ceramide accumulation. Liver insulin sensitivity was improved in Plin5-/- mice, indicating tissue-specific effects of PLIN5 on insulin action. We conclude that PLIN5 plays a critical role in coordinating skeletal muscle triacylglycerol metabolism, which impacts sphingolipid metabolism, and is requisite for the maintenance of skeletal muscle insulin action. © 2014 The Authors.

Cellular localization and associations of the major lipolytic proteins in human skeletal muscle at rest and during exercise

Lipolysis involves the sequential breakdown of fatty acids from triacylglycerol and is increased during energy stress such as exercise. Adipose triglyceride lipase (ATGL) is a key regulator of skeletal muscle lipolysis and perilipin (PLIN) 5 is postulated to be an important regulator of ATGL action of muscle lipolysis. Hence, we hypothesized that non-genomic regulation such as cellular localization and the interaction of these key proteins modulate muscle lipolysis during exercise. PLIN5, ATGL and CGI-58 were highly (>60%) colocated with Oil Red O (ORO) stained lipid droplets. PLIN5 was significantly colocated with ATGL, mitochondria and CGI-58, indicating a close association between the key lipolytic effectors in resting skeletal muscle. The colocation of the lipolytic proteins, their independent association with ORO and the PLIN5/ORO colocation were not altered after 60 min of moderate intensity exercise. Further experiments in cultured human myocytes showed that PLIN5 colocation with ORO or mitochondria is unaffected by pharmacological activation of lipolytic pathways. Together, these data suggest that the major lipolytic proteins are highly expressed at the lipid droplet and colocate in resting skeletal muscle, that their localization and interactions appear to remain unchanged during prolonged exercise, and, accordingly, that other post-translational mechanisms are likely regulators of skeletal muscle lipolysis.

Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes

Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.

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.

A computational search for lipases that can preferentially hydrolyze long-chain omega-3 fatty acids from fish oil triacylglycerols

Consumption of long-chain omega-3 fatty acids is known to decrease the risk of major cardiovascular events. Lipases, a class of triacylglycerol hydrolases, have been extensively tested to concentrate omega-3 fatty acids from fish oils, under mild enzymatic conditions. However, no lipases with preference for omega-3 fatty acids selectivity have yet been discovered or developed. In this study we performed an exhaustive computational study of substrate-lipase interactions by docking, both covalent and non-covalent, for 38 lipases with a large number of structured triacylglycerols containing omega-3 fatty acids. We identified some lipases that have potential to preferentially hydrolyze omega-3 fatty acids from structured triacylglycerols. However omega-3 fatty acid preferences were found to be modest. Our study provides an explanation for absence of reports of lipases with omega-3 fatty acid hydrolyzing ability and suggests methods for developing these selective lipases.

Lipid profiles, in vitro digestion and oxidative stability of mutton bird oil

The lipid profile, in vitro digestion and oxidative stability of mutton bird oil were investigated. Wax ester, triacylglycerol and sterol were the major lipids present as determined using capillary chromatography with flame ionisation detector (Iatroscan). Fatty acid analysis by gas chromatography (GC) showed that wax esters had a higher total omega-3 fatty acids content including EPA, DPA and DHA than TAGs (31 % and 24 %, respectively). In TAGs, 13C nuclear magnetic resonance (NMR) data showed that EPA was statistically positioned at sn-1,3 and sn-2, while DHA was preferentially at sn-2. In vitro digestion using porcine pancreatic lipase resulted in 75 % of TAG and 10 % wax ester hydrolysis in 120 min. As reflected in the measured conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) values during accelerated oxidation at 60 °C for 5 days, the oil was relatively stable against oxidation considering its high omega-3 content.