Synergistic Effect of Vespa Amino Acid Mixture on Lipolysis in Rat Adipocytes

The effect of Vespa amino acid mixture (VAAM) on the release of lipolytic products was examined in isolated rat adipocytes. Concentrations of 112.5 to 225 ppm of VAAM showed significantly greater release of non-esterified fatty acids (NEFA) and glycerol than the same concentrations of casein amino acid mixture (CAAM). The integrated relative release of NEFA and glycerol was lower in response to individual administration of amino acids comprising VAAM than to VAAM itself. Further, amino acids mixtures deficient in a single amino acid comprising VAAM showed significantly lower release of lipolytic products than VAAM. These data suggest that the synergistic effect of VAAM on the release of lipolytic products is a function of concurrent exposure to the unique composition of amino acids found in VAAM as compared to the effect of exposure to the same individual un-mixed amino acids or to a mixture lacking one of the amino acids comprising VAAM.     

Anti‐lipolytic Effects of Insulin in African American and White Prepubertal Boys

Objective: Relative to whites, African Americans have lower circulating triglycerides (TG) and greater highdensity lipoprotein cholesterol. The metabolic basis for this difference is not known. This study was conducted to test the hypothesis that insulin‐induced suppression of free fatty acids (FFA) results in lower serum TG in African American versus white prepubertal children. Research Methods and Procedures: Insulin, FFA, and TG were determined at baseline and during a frequently sampled, intravenous glucose tolerance test in eight African American and eight white prepubertal males pair‐matched for whole‐body insulin sensitivity. Results: Baseline TG was lower in African Americans (0.43 ± 0.10 vs. 0.79 ± 0.37 mM/L; mean ± SD; p < 0.01). African Americans had higher peak insulin (218 ± 102 vs. 100 ± 30 pM/L; mean ± SD; p < 0.01) and a greater acute insulin response (9282 ± 4272 vs. 4230 ± 1326 pM/L × 10 minutes; mean ± SD; p < 0.05). FFA and TG values determined at the FFA nadir were lower in African Americans (0.26 ± 0.02 vs. 0.30 ± 0.03 mEq/L; mean ± SD; p < 0.01 for FFA nadir and 0.49 ± 0.07 vs. 0.77 ± 0.33 mM/L; mean ± SD; p < 0.05 for TG). Among all subjects, FFA nadir was correlated with peak insulin (r = ?0.54; p < 0.05). After adjusting for FFA nadir, neither baseline nor postchallenge TG differed with ethnicity (p = 0.073 and 0.192, respectively). The ethnic difference in FFA nadir disappeared after adjusting for peak insulin (p = 0.073). Discussion: These data suggest that hyperinsulinemiainduced suppression of FFA among African Americans is a determinant of lower TG in this group.     

Neuropeptides,Including Neuropeptide Y and Melanocortins,Mediate Lipolysis in Murine Adipocytes

Objective: To determine whether key appetite‐regulating neuropeptides such as melanin‐concentrating hormone (MCH), neuropeptide Y (NPY), and α‐melanocyte—stimulating hormone (α‐MSH), which are known to mediate energy balance through centrally mediated pathways, also have direct acute effects on the lipolytic activity of murine adipocytes. Research Methods and Procedures: Fully differentiated 3T3‐L1 adipocytes serum starved overnight in Dulbecco's modified Eagle medium containing 2% bovine serum albumin or freshly isolated mouse adipocytes were incubated for up to 2 hours in the absence and presence of 100 nM each of NPY, MCH, α‐MSH, the melanocortin receptor agonist MTII, or isoproterenol as a control. Free fatty acids secreted into the incubation medium were measured using a commercially available nonesterified fatty acid C test kit. Results: Treatment of 3T3‐L1 cells with 100 nM NPY decreased basal free fatty acid secretion (basal, 0.006 ± 0.001 vs. NPY, 0.001 ± 0.0003 nM at 90 minutes; p < 0.05), whereas both α‐MSH and MTII stimulated up to a 7‐fold increase in free fatty acid release (MTII, 0.238 ± 0.004 vs. basal, 0.024 ± 0.002 nM at 2 hours; p < 0.05; and α‐MSH, 0.22 ± 0.005 vs. basal, 0.04 ± 0.003 nM at 2 hours; p < 0.05). Treatment with 100 nM MCH had no effect on basal free fatty acid release or on α‐MSH—induced lipolysis during concurrent treatment. Conversely, concurrent treatment with 100 nM NPY dramatically inhibited (by ～90%) α‐MSH—induced lipolysis. Similar treatment of freshly isolated mouse adipocytes showed virtually identical results. Discussion: In addition to their centrally mediated actions, appetite‐regulating neuropeptides modulate adipose tissue mass through direct peripheral effects. Systemic administration of pharmacological agents altering the effects of these neuropeptides may form the basis of future obesity therapies. Thus, some of these agents will likely have direct effects on adipocytes that may serve to alter their therapeutic effectiveness.     

Regulation of Acetyl CoA Carboxylase and Carnitine Palmitoyl Transferase‐1 in Rat Adipocytes

Objective: Acetyl CoA carboxylase (ACC) is a key enzyme in energy balance. It controls the synthesis of malonyl‐CoA, an allosteric inhibitor of carnitine palmitoyltransferase‐1 (CPT‐I). CPT‐I is the gatekeeper of free fatty acid (FFA) oxidation. To test the hypothesis that both enzymes play critical roles in regulation of FFA partitioning in adipocytes, we compared enzyme mRNA expression and specific activity from fed, fasted, and diabetic rats. Research Methods and Procedures: Direct effects of nutritional state, insulin, and FFAs on CPT‐I and ACC mRNA expression were assessed in adipocytes, liver, and cultured adipose tissue explants. We also determined FFA partitioning in adipocytes from donors exposed to different nutritional conditions. Results: CPT‐I mRNA and activity decreased in adipocytes but increased in liver in response to fasting. ACC mRNA and activity decreased in both adipocytes and liver during fasting. These changes were not caused directly by fasting‐associated changes in plasma insulin and FFA concentrations because insulin suppressed CPT‐I mRNA and did not affect ACC mRNA in vitro, whereas exogenous oleate had no effect on either. Despite the decrease in adipocyte CPT‐I mRNA and specific activity, CO₂ production from endogenous FFAs increased, suggesting increased FFA transport through CPT‐I for β‐oxidation. Discussion: Stimulation of FFA transport through CPT‐I occurs in both tissues, but CPT‐I mRNA and specific activity correlate with FFA transport in liver and not in adipocytes. We conclude that the mechanism responsible for increasing FFA oxidation in adipose tissue during fasting involves mainly allosteric regulation, whereas altered gene expression may play a central role in the liver.     

Inflammatory profile in X‐linked adrenoleukodystrophy patients: Understanding disease progression

X‐linked adrenoleukodystrophy (X‐ALD) is an inherited disease characterized by progressive inflammatory demyelization in the brain, adrenal insufficiency, and an abnormal accumulation of very long chain fatty acids (VLCFA) in tissue and body fluids. Considering that inflammation might be involved in pathophysiology of X‐ALD, we aimed to investigate pro‐ and anti‐inflammatory cytokines in plasma from three different male phenotypes (CCER, AMN, and asymptomatic individuals). Our results showed that asymptomatic patients presented increased levels of pro‐inflammatory cytokines IL‐1β, IL‐2, IL‐8, and TNF‐α and the last one was also higher in AMN phenotype. Besides, asymptomatic patients presented higher levels of anti‐inflammatory cytokines IL‐4 and IL‐10. AMN patients presented higher levels of IL‐2, IL‐5, and IL‐4. We might hypothesize that inflammation in X‐ALD is related to plasmatic VLCFA concentration, since there were positive correlations between C26:0 plasmatic levels and pro‐inflammatory cytokines in asymptomatic and AMN patients and negative correlation between anti‐inflammatory cytokine and C24:0/C22:0 ratio in AMN patients. The present work yields experimental evidence that there is an inflammatory imbalance associated Th1, (IL‐2, IL‐6, and IFN‐γ), Th2 (IL‐4 and IL‐10), and macrophages response (TNF‐α and IL‐1β) in the periphery of asymptomatic and AMN patients, and there is correlation between VLCFA plasmatic levels and inflammatory mediators in X‐ALD. Furthermore, we might also speculate that the increase of plasmatic cytokines in asymptomatic patients could be considered an early biomarker of brain damage and maybe also a predictor of disease progression.     

Dietary n‐3 long‐chain polyunsaturated fatty acids modify phosphoenolpyruvate carboxykinase activity and lipid synthesis from glucose in adipose tissue of rats fed a high‐sucrose diet

Long‐chain polyunsaturated n‐3 fatty acids (n‐3 LCPUFAs) have hypolipidemic effects and modulate intermediary metabolism to prevent or reverse insulin resistance in a way that is not completely elucidated. Here, effects of these fatty acids on the lipid profile, phosphoenolpyruvate carboxykinase (PEPCK) activity, lipid synthesis from glucose in epididymal adipose tissue (Ep‐AT) and liver were investigated. Male rats were fed a high‐sucrose diet (SU diet), containing either sunflower oil or a mixture of sunflower and fish oil (SU–FO diet), and the control group was fed a standard diet. After 13 weeks, liver, adipose tissue and blood were harvested and analysed. The dietary n‐3 LCPUFAs prevented sucrose‐induced increase in adiposity and serum free fat acids, serum and hepatic triacylglycerol and insulin levels. Furthermore, these n‐3 LCPUFAs decreased lipid synthesis from glucose and increased PEPCK activity in the Ep‐AT of rats fed the SU–FO diet compared to those fed the SU diet, besides reducing lipid synthesis from glucose in hepatic tissue. Thus, the inclusion of n‐3 LCPUFAs in the diet may be beneficial for the prevention or attenuation of dyslipidemia and insulin resistance, and for reducing the risk of related chronic diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

Climate warming is predicted to reduce omega‐3, long‐chain,polyunsaturated fatty acid production in phytoplankton

Phytoplankton are the main source of energy and omega‐3 (n‐3) long‐chain essential fatty acids (EFA) in aquatic ecosystems. Their growth and biochemical composition are affected by surrounding environmental conditions, including temperature, which continues to increase as a result of climate warming. Increasing water temperatures may negatively impact the production of EFA by phytoplankton through the process of homeoviscous adaptation. To investigate this, we conducted an exploratory data synthesis with 952 fatty acid (FA) profiles from six major groups of marine and freshwater phytoplankton. Temperature was strongly correlated with a decrease in the proportion of n‐3 long‐chain polyunsaturated FA (LC‐PUFA) and an increase in omega‐6 FA and saturated FA. Based on linear regression models, we predict that global n‐3 LC‐PUFA production will be reduced by 8.2% for eicosapentaenoic acid (EPA) and 27.8% for docosahexaenoic acid (DHA) with an increase in water temperature of 2.5 °C. Using a previously published estimate of the global production of EPA by diatoms, which contribute to most of the world's supply of EPA, we predict a loss of 14.2 Mt of EPA annually as a result of ocean warming. The n‐3 LC‐PUFA are vitally important for an array of key physiological functions in aquatic and terrestrial organisms, and these FA are mainly produced by phytoplankton. Therefore, reduced production of these EFA, as a consequence of climate warming, is predicted to negatively affect species that depend on these compounds for optimum physiological function. Such profound changes in the biochemical composition of phytoplankton cell membranes can lead to cascading effects throughout the world's ecosystems.     

Alteration of fatty acid chain length of Chlamydomonas reinhardtii by simultaneous expression of medium‐chain‐specific thioesterase and acyl carrier protein

Biofuel from fatty acids with chain lengths of 8–15 (C8–C15) have properties similar to those of conventional diesel and jet fuels, thus, can save time and reduce costs for the refurbishment of engines and maintenance of oiling facilities. Most oil‐producing algae yield C16–C18 fatty acids; however, the manipulation of algae using genetic engineering is a promising approach to obtain C8–C15 fatty acids. The introduction of a medium‐chain‐specific thioesterase (TE) is expected to effectively alter algae to produce medium‐chain fatty acids (MCFAs). TE is the main determinant of fatty acid chain length as it releases fatty acids from the acyl carrier protein (ACP) in the fatty acid elongation cycle. In a previous study, the introduction of heterologous C8–C12‐specific TEs into Chlamydomonas reinhardtii did not increase the yield of MCFAs. This effect was attributed to a low affinity of the heterologous TEs to C. reinhardtii ACP. Therefore, we introduced both the C10–C14‐specific TE gene and the ACP gene from the land plant Cuphea lanceolata into C. reinhardtii. We measured free fatty acids (FFAs) and triacylglycerols (TAGs) in the transformants using liquid chromatography–mass spectrometry. The production of C12:0 and C14:0, chain length 12 and 14 without unsaturation, FFAs was not significantly increased in any of the tested strains. However, we found a slight but significant increase in TAG‐containing MCFAs in both TE only and TE–ACP transformants. The increased production rate of C14:0‐containing TAGs ranged from 1.25‐ to 1.58‐fold, indicating the ability of medium‐chain‐specific TE to increase MCFAs. These results suggest that the selection of specific TEs is important when modifying eukaryotic algae to produce MCFAs.     

Unique coenzyme binding mode of hyperthermophilic archaeal sn‐glycerol‐1‐phosphate dehydrogenase from Pyrobaculum calidifontis

A gene encoding an sn‐glycerol‐1‐phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V_max) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2’ of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild‐type enzyme with NADPH, which suggests that the biased interactions around the C2’‐phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)‐dependent dehydrogenases. Proteins 2016; 84:1786–1796. © 2016 Wiley Periodicals, Inc.     

AKR2A interacts with KCS1 to improve VLCFAs contents and chilling tolerance of Arabidopsis thaliana

Arabidopsis thaliana AKR2A plays an important role in plant responses to cold stress. However, its exact function in plant resistance to cold stress remains unclear. In the present study, we found that the contents of very long‐chain fatty acids (VLCFAs) in akr2a mutants were decreased, and the expression level of KCS1 was also reduced. Overexpression of KCS1 in the akr2a mutants could enhance VLCFAs contents and chilling tolerance. Yeast‐2‐hybrid and bimolecular fluorescence complementation (BIFC) results showed that the transmembrane motif of KCS1 interacts with the PEST motif of AKR2A both in vitro and in vivo. Overexpression of KCS1 in akr2a mutants rescued akr2a mutant phenotypes, including chilling sensitivity and a decrease of VLCFAs contents. Moreover, the transgenic plants co‐overexpressing AKR2A and KCS1 exhibited a greater chilling tolerance than the plants overexpressing AKR2A or KCS1 alone, as well as the wild‐type. AKR2A knockdown and kcs1 knockout mutants showed the worst performance under chilling conditions. These results indicate that AKR2A is involved in chilling tolerance via an interaction with KCS1 to affect VLCFA biosynthesis in Arabidopsis.