Shark liver oil supplementation enriches endogenous plasmalogens and reduces markers of dyslipidemia and inflammation.

Plasmalogens are membrane glycerophospholipids with diverse biological functions. Reduced plasmalogen levels have been observed in metabolic diseases; hence, increasing their levels might be beneficial in ameliorating these conditions. Shark liver oil (SLO) is a rich source of alkylglycerols that can be metabolized into plasmalogens. This study was designed to evaluate the impact of SLO supplementation on endogenous plasmalogen levels in individuals with features of metabolic disease. In this randomized, double-blind, placebo-controlled cross-over study, the participants (10 overweight or obese males) received 4-g Alkyrol® (purified SLO) or placebo (methylcellulose) per day for 3 weeks followed by a 3-week washout phase and were then crossed over to 3 weeks of the alternate placebo/Alkyrol® treatment. SLO supplementation led to significant changes in plasma and circulatory white blood cell lipidomes, notably increased levels of plasmalogens and other ether lipids. In addition, SLO supplementation significantly decreased the plasma levels of total free cholesterol, triglycerides, and C-reactive protein. These findings suggest that SLO supplementation can enrich plasma and cellular plasmalogens and this enrichment may provide protection against obesity-related dyslipidemia and inflammation.

Oral Supplementation of an Alkylglycerol Mix Comprising Different Alkyl Chains Effectively Modulates Multiple Endogenous Plasmalogen Species in Mice.

Plasmalogens or alkenylphospholipids are a sub-class of glycerophospholipids with numerous biological functions and are thought to have protective effects against metabolic disease. Dietary supplementation with alkylglycerols (AKGs) has been shown to increase endogenous plasmalogen levels, however effective modulation of different molecular plasmalogen species has not yet been demonstrated. In this study, the effects of an orally-administered AKG mix (a mixture of chimyl, batyl and selachyl alcohol at a 1:1:1 ratio) on plasma and tissue lipids, including plasmalogens, was evaluated. Mice on a Western-type diet were treated with either an AKG mix or vehicle (lecithin) for 1, 2, 4, 8 and 12 weeks. Treatment with the AKG mix significantly increased the total plasmalogen content of plasma, liver and adipose tissue as a result of elevations in multiple plasmalogen species with different alkenyl chains. Alkylphospholipids, the endogenous precursors of plasmalogens, showed a rapid and significant increase in plasma, adipose tissue, liver and skeletal muscle. A significant accumulation of alkyl-diacylglycerol and lyso-ether phospholipids was also observed in plasma and tissues. Additionally, the dynamics of plasmalogen-level changes following AKG mix supplementation differed between tissues. These findings indicate that oral supplementation with an AKG mix is capable of upregulating and maintaining stable expression of multiple molecular plasmalogen species in circulation and tissues.

High placental inositol content associated with suppressed pro-adipogenic effects of maternal glycaemia in offspring: the GUSTO cohort.

BACKGROUND/OBJECTIVES: Maternal glycaemia promotes fetal adiposity. Inositol, an insulin sensitizer, has been trialled for gestational diabetes prevention. The placenta has been implicated in how maternal hyperglycaemia generates fetal pathophysiology, but no studies have examined whether placental inositol biology is altered with maternal hyperglycaemia, nor whether such alterations impact fetal physiology. We aimed to investigate whether the effects of maternal glycaemia on offspring birthweight and adiposity at birth differed across placental inositol levels. METHODS: Using longitudinal data from the Growing Up in Singapore Towards healthy Outcomes cohort, maternal fasting glucose (FPG) and 2-hour plasma glucose (2hPG) were obtained in pregnant women by a 75-g oral glucose tolerance test around 26 weeks' gestation. Relative placental inositol was quantified by liquid chromatography-mass spectrometry. Primary outcomes were birthweight (n = 884) and abdominal adipose tissue (AAT) volumes measured by neonatal MRI scanning in a subset (n = 262) of term singleton pregnancies. Multiple linear regression analyses were performed. RESULTS: Placental inositol was lower in those with higher 2hPG, no exposure to tobacco smoke antenatally, with vaginal delivery and shorter gestation. Positive associations of FPG with birthweight (adjusted ß [95% CI] 164.8 g [109.1, 220.5]) and AAT (17.3 ml [11.9, 22.6] per mmol glucose) were observed, with significant interactions between inositol tertiles and FPG in relation to these outcomes (p < 0.05). Stratification by inositol tertiles showed that each mmol/L increase in FPG was associated with increased birthweight and AAT volume among cases within the lowest (birthweight = 174.2 g [81.2, 267.2], AAT = 21.0 ml [13.1, 28.8]) and middle inositol tertiles (birthweight = 202.0 g [103.8, 300.1], AAT = 19.7 ml [9.7, 29.7]). However, no significant association was found among cases within the highest tertile (birthweight = 81.0 g [-21.2, 183.2], AAT = 0.8 ml [-8.4, 10.0]). CONCLUSIONS: High placental inositol may protect the fetus from the pro-adipogenic effects of maternal glycaemia. Studies are warranted to investigate whether prenatal inositol supplementation can increase placental inositol and reduce fetal adiposity.

Krill Oil Has Different Effects on the Plasma Lipidome Compared with Fish Oil Following 30 Days of Supplementation in Healthy Women: A Randomized Controlled and Crossover Study.

This is a follow-up of our previous postprandial study and it focused on the plasma lipidomic responses to 30 days of krill oil (KO) versus fish oil (FO) supplementations in healthy women. Eleven women (aged 18-50 years) consumed KO or FO for 30 days in a randomized, cross-over study, with at least a four-week washout period between supplementations. The daily supplements provided 1.27 g/day of long-chain (LC) omega-3 polyunsaturated fatty acids (PUFA) from KO (containing 0.76 g eicosapentaenoic acid (EPA), 0.42 g docosahexaenoic acid (DHA)) and 1.44 g/day from FO (containing 0.79 g EPA, 0.47 g DHA). Fasting plasma samples at days 0, 15, and 30 were analyzed using gas chromatography and liquid chromatography electrospray ionisation-tandem mass spectrometry. KO resulted in a significantly greater relative area under the curve (relAUC) for plasma EPA after 30 days. Lipidomic analysis showed that 26 of 43 lipid molecular species had a significantly greater relAUC in the KO group, while 17/43 showed a significantly lower relAUC compared with the FO group. More than 38% of the lipids species which increased more following KO contained omega-3 PUFA, while where FO was greater than KO, only 12% contained omega-3 PUFA. These data show that KO and FO do not have equivalent effects on the plasma lipidome.

Insulin signaling requires glucose to promote lipid anabolism in adipocytes.

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.

Short-term inhibition of autophagy benefits pancreatic ß-cells by augmenting ether lipids and peroxisomal function, and by countering depletion of n-3 polyunsaturated fatty acids after fat-feeding.

OBJECTIVE: Investigations of autophagy in ß-cells have usually focused on its homeostatic function. More dynamic roles in inhibiting glucose-stimulated insulin secretion (GSIS), potentially involving remodelling of cellular lipids, have been suggested from in vitro studies but not evaluated in vivo. METHODS: We employed temporally-regulated deletion of the essential autophagy gene, Atg7, in ß-cells. Mice were fed chow or high-fat diets (HFD), in conjunction with deletion of Atg7 for the last 3 weeks (short-term model) or 9 weeks (long-term model). Standard in vivo metabolic phenotyping was undertaken, and 450 lipid species in islets quantified ex vivo using mass spectroscopy (MS). MIN6 cells were also employed for lipidomics and secretory interventions. RESULTS: ß-cell function was impaired by inhibiting autophagy in the longer-term, but conversely improved by 3-week deletion of Atg7, specifically under HFD conditions. This was accompanied by augmented GSIS ex vivo. Surprisingly, the HFD had minimal effect on sphingolipid and neutral lipid species, but modulated >100 phospholipids and ether lipids, and markedly shifted the profile of polyunsaturated fatty acid (PUFA) sidechains from n3 to n6 forms. These changes were partially countered by Atg7 deletion, consistent with an accompanying upregulation of the PUFA elongase enzyme, Elovl5. Loss of Atg7 separately augmented plasmalogens and alkyl lipids, in association with increased expression of Lonp2, a peroxisomal chaperone/protease that facilitates maturation of ether lipid synthetic enzymes. Depletion of PUFAs and ether lipids was also observed in MIN6 cells chronically exposed to oleate (more so than palmitate). GSIS was inhibited by knocking down Dhrs7b, which encodes an enzyme of peroxisomal ether lipid synthesis. Conversely, impaired GSIS due to oleate pre-treatment was selectively reverted by Dhrs7b overexpression. CONCLUSIONS: A detrimental increase in n6:n3 PUFA ratios in ether lipids and phospholipids is revealed as a major response of ß-cells to high-fat feeding. This is partially reversed by short-term inhibition of autophagy, which results in compensatory changes in peroxisomal lipid metabolism. The short-term phenotype is linked to improved GSIS, in contrast to the impairment seen with the longer-term inhibition of autophagy. The balance between these positive and negative inputs could help determine whether ß-cells adapt or fail in response to obesity.

Differential plasma postprandial lipidomic responses to krill oil and fish oil supplementations in women: A randomized crossover study.

OBJECTIVES: There is no convincing evidence that krill oil (KO) consumption results in a higher incorporation of long chain ω-3 polyunsaturated fatty acids into blood lipid fractions than fish oil (FO). This study examined the postprandial plasma lipidomic responses to KO supplementation compared with FO supplementation in healthy women. METHODS: Ten women (aged 18-45 y) consumed a high-fat (15 g of olive oil) breakfast, supplemented with 5 g of KO or FO in a randomized crossover study with a minimum 7-d washout period between the supplementations. Plasma samples collected at the fasting state and at 3 and 5 h postprandially were analyzed using liquid chromatography electrospray ionization-tandem mass spectrometry. RESULTS: After the supplementations, 5 out of 34 lipid classes or subclasses had significantly greater concentrations from KO compared with FO. There were 27 molecular species including 5 ether-phospholipid species, out of a total of 701, which had significant differences between supplementations in the postprandial period. Eicosapentaenoic acid and docosahexaenoic acid from KO were preferentially partitioned toward phospholipid molecular species, whereas eicosapentaenoic acid and docosahexaenoic acid from FO were preferentially partitioned toward neutral lipids.

Plasmalogens: A potential therapeutic target for neurodegenerative and cardiometabolic disease.

Plasmalogens are a class of membrane glycerophospholipids with unique properties. They contain a vinyl-ether linked alkyl chain at the sn-1 position of the glycerol backbone and, typically, a polyunsaturated fatty acyl chain at the sn-2 position. Plasmalogens are critical for human health and have established roles in neuronal development, the immune response and as endogenous antioxidants. However, the mechanistic bases of these and other biological functions of plasmalogens are not well defined. Lipidomic studies have characterised reduced levels of plasmalogens in a number of disease states, including neurodegenerative and cardiometabolic disease, highlighting the potential of plasmalogen modulation as a therapeutic strategy. A number of approaches have been proposed to upregulate plasmalogen levels in different clinical settings; these include dietary intervention with inositol or the naturally occurring metabolic precursors known as alkylglycerols. Plasmalogen modulation has been utilised in both preclinical and clinical studies to prevent onset and/or attenuate progression of neurodegenerative diseases, atherosclerosis, insulin resistance and hepatosteatosis. These studies are providing new insight into the mechanistic role of plasmalogens in disease and their therapeutic potential. In this review, we will examine the strategies for plasmalogen modulation and recent progress toward therapeutic applications with a focus on neurodegenerative and cardiometabolic disease.

Drug induced exocytosis of glycogen in Pompe disease.

Pompe disease is caused by a deficiency in the lysosomal enzyme α-glucosidase, and this leads to glycogen accumulation in the autolysosomes of patient cells. Glycogen storage material is exocytosed at a basal rate in cultured Pompe cells, with one study showing up to 80% is released under specific culture conditions. Critically, exocytosis induction may reduce glycogen storage in Pompe patients, providing the basis for a therapeutic strategy whereby stored glycogen is redirected to an extracellular location and subsequently degraded by circulating amylases. The focus of the current study was to identify compounds capable of inducing rapid glycogen exocytosis in cultured Pompe cells. Here, calcimycin, lysophosphatidylcholine and α-l-iduronidase each significantly increased glycogen exocytosis compared to vehicle-treated controls. The most effective compound, calcimycin, induced exocytosis through a Ca2+-dependent mechanism, although was unable to release a pool of vesicular glycogen larger than the calcimycin-induced exocytic pore. There was reduced glycogen release from Pompe compared to unaffected cells, primarily due to increased granule size in Pompe cells. Drug induced exocytosis therefore shows promise as a therapeutic approach for Pompe patients but strategies are required to enhance the release of large molecular weight glycogen granules.

Plasmalogen modulation attenuates atherosclerosis in ApoE- and ApoE/GPx1-deficient mice.

BACKGROUND AND AIM: We previously reported a negative association of circulating plasmalogens (phospholipids with proposed atheroprotective properties) with coronary artery disease. Plasmalogen modulation was previously demonstrated in animals but its effect on atherosclerosis was unknown. We assessed the effect of plasmalogen enrichment on atherosclerosis of murine models with differing levels of oxidative stress. METHODS AND RESULTS: Six-week old ApoE- and ApoE/glutathione peroxidase-1 (GPx1)-deficient mice were fed a high-fat diet with/without 2% batyl alcohol (precursor to plasmalogen synthesis) for 12 weeks. Mass spectrometry analysis of lipids showed that batyl alcohol supplementation to ApoE- and ApoE/GPx1-deficient mice increased the total plasmalogen levels in both plasma and heart. Oxidation of plasmalogen in the treated mice was evident from increased level of plasmalogen oxidative by-product, sn-2 lysophospholipids. Atherosclerotic plaque in the aorta was reduced by 70% (P = 5.69E-07) and 69% (P = 2.00E-04) in treated ApoE- and ApoE/GPx1-deficient mice, respectively. A 40% reduction in plaque (P = 7.74E-03) was also seen in the aortic sinus of only the treated ApoE/GPx1-deficient mice. Only the treated ApoE/GPx1-deficient mice showed a decrease in VCAM-1 staining (-28%, P = 2.43E-02) in the aortic sinus and nitrotyrosine staining (-78%, P = 5.11E-06) in the aorta. CONCLUSION: Plasmalogen enrichment via batyl alcohol supplementation attenuated atherosclerosis in ApoE- and ApoE/GPx1-deficient mice, with a greater effect in the latter group. Plasmalogen enrichment may represent a viable therapeutic strategy to prevent atherosclerosis and reduce cardiovascular disease risk, particularly under conditions of elevated oxidative stress and inflammation.

Statin action favors normalization of the plasma lipidome in the atherogenic mixed dyslipidemia of MetS: potential relevance to statin-associated dysglycemia.

The impact of statin treatment on the abnormal plasma lipidome of mixed dyslipidemic patients with metabolic syndrome (MetS), a group at increased risk of developing diabetes, was evaluated. Insulin-resistant hypertriglyceridemic hypertensive obese males (n = 12) displaying MetS were treated with pitavastatin (4 mg/day) for 180 days; healthy normolipidemic age-matched nonobese males (n = 12) acted as controls. Statin treatment substantially normalized triglyceride (-41%), remnant cholesterol (-55%), and LDL-cholesterol (-39%), with minor effect on HDL-cholesterol (+4%). Lipidomic analysis, normalized to nonHDL-cholesterol in order to probe statin-induced differences in molecular composition independently of reduction in plasma cholesterol, revealed increment in 132 of 138 lipid species that were subnormal at baseline and significantly shifted toward the control group on statin treatment. Increment in alkyl- and alkenylphospholipids (plasmalogens) was prominent, and consistent with significant statin-induced increase in plasma polyunsaturated fatty acid levels. Comparison of the statin-mediated lipidomic changes in MetS with the abnormal plasma lipidomic profile characteristic of prediabetes and T2D in the Australian Diabetes, Obesity, and Lifestyle Study and San Antonio Family Heart Study cohorts by hypergeometric analysis revealed a significant shift toward the lipid profile of controls, indicative of a marked trend toward a normolipidemic phenotype. Pitavastatin attenuated the abnormal plasma lipidome of MetS patients typical of prediabetes and T2D.

Dietary sphingomyelin lowers hepatic lipid levels and inhibits intestinal cholesterol absorption in high-fat-fed mice.

Controlling intestinal lipid absorption is an important strategy for maintaining lipid homeostasis. Accumulation of lipids in the liver is a major risk factor for metabolic syndrome and nonalcoholic fatty liver disease. It is well-known that sphingomyelin (SM) can inhibit intestinal cholesterol absorption. It is, however, unclear if dietary SM also lowers liver lipid levels. In the present study (i) the effect of pure dietary egg SM on hepatic lipid metabolism and intestinal cholesterol absorption was measured with [(14)C]cholesterol and [(3)H]sitostanol in male C57BL/6 mice fed a high-fat (HF) diet with or without 0.6% wt/wt SM for 18 days; and (ii) hepatic lipid levels and gene expression were determined in mice given a HF diet with or without egg SM (0.3, 0.6 or 1.2% wt/wt) for 4 weeks. Mice supplemented with SM (0.6% wt/wt) had significantly increased fecal lipid and cholesterol output and reduced hepatic [(14)C]cholesterol levels after 18 days. Relative to HF-fed mice, SM-supplemented HF-fed mice had significantly lower intestinal cholesterol absorption (-30%). Liver weight was significantly lower in the 1.2% wt/wt SM-supplemented mice (-18%). Total liver lipid (mg/organ) was significantly reduced in the SM-supplemented mice (-33% and -40% in 0.6% wt/wt and 1.2% wt/wt SM, respectively), as were triglyceride and cholesterol levels. The reduction in liver triglycerides was due to inactivation of the LXR-SREBP-1c pathway. In conclusion, dietary egg SM has pronounced hepatic lipid-lowering properties in mice maintained on an obesogenic diet.

Lipidomic profiling of chylomicron triacylglycerols in response to high fat meals.

Using lipidomic methodologies the impact that meal lipid composition and metabolic syndrome (MetS) exerts on the postprandial chylomicron triacylglycerol (TAG) response was examined. Males (9 control; 11 MetS) participated in a randomised crossover trial ingesting two high fat breakfast meals composed of either dairy-based foods or vegetable oil-based foods. The postprandial lipidomic molecular composition of the TAG in the chylomicron-rich (CM) fraction was analysed with tandem mass spectrometry coupled with liquid chromatography to profile CM TAG species and targeted TAG regioisomers. Postprandial CM TAG concentrations were significantly lower after the dairy-based foods compared with the vegetable oil-based foods for both control and MetS subjects. The CM TAG response to the ingested meals involved both significant and differential depletion of TAG species containing shorter- and medium-chain fatty acids (FA) and enrichment of TAG molecular species containing C16 and C18 saturated, monounsaturated and diunsaturated FA. Furthermore, there were significant changes in the TAG species between the food TAG and CM TAG and between the 3- and 5-h postprandial samples for the CM TAG regioisomers. Unexpectedly, the postprandial CM TAG concentration and CM TAG lipidomic responses did not differ between the control and MetS subjects. Lipidomic analysing of CM TAG molecular species revealed dynamic changes in the molecular species of CM TAG during the postprandial phase suggesting either preferential CM TAG species formation and/or clearance.

Consumption of a high-fat diet, but not regular endurance exercise training, regulates hypothalamic lipid accumulation in mice.

Obesity is characterised by increased storage of fatty acids in an expanded adipose tissue mass and in peripheral tissues such as the skeletal muscle and liver, where it is associated with the development of insulin resistance. Insulin resistance also develops in the central nervous system with high-fat feeding. The capacity for hypothalamic cells to accumulate/store lipids, and the effects of obesity remain undefined. The aims of this study were (1) to examine hypothalamic lipid content in mice with increased dietary fat intake and in obese ob/ob mice fed a low-fat diet, and (2) to determine whether endurance exercise training could reduce hypothalamic lipid accumulation in high-fat fed mice. Male C57BL/6 mice were fed a low- (LFD) or high-fat diet (HFD) for 12 weeks; ob/ob mice were maintained on a chow diet. HFD-exercise (HFD-ex) mice underwent 12 weeks of high-fat feeding with 6 weeks of treadmill exercise training (increasing from 30 to 70 min day(-1)). Hypothalamic lipids were assessed by unbiased mass spectrometry. The HFD increased body mass and hepatic lipid accumulation, and induced glucose intolerance, while the HFD-ex mice had reduced body weight and improved glucose tolerance. A total of 335 lipid molecular species were identified and quantified. Lipids known to induce insulin resistance, including ceramide (22%↑), diacylglycerol (25%↑), lysophosphatidylcholine (17%↑), cholesterol esters (60%↑) and dihexosylceramide (33%↑), were increased in the hypothalamus of HFD vs. LFD mice. Hypothalamic lipids were unaltered with exercise training and in the ob/ob mice, suggesting that obesity per se does not alter hypothalamic lipids. Overall, hypothalamic lipid accumulation is regulated by dietary lipid content and is refractory to change with endurance exercise training.

Hydrogenated phosphatidylcholine supplementation reduces hepatic lipid levels in mice fed a high-fat diet.

The ability of the fatty acid composition of dietary phosphatidylcholine (PC) to affect hepatic lipid levels was investigated in C57BL/6 mice (n=8-10 per group) by feeding: (1) a high-fat semi-purified diet (HF), (2) HF diet supplemented with 1.25 wt% soy PC (SPC), (3) HF with 1.25 wt% hydrogenated soy PC (SPCH), (4) HF with 1.25 wt% egg PC (EPC), and (5) HF with 1.25 wt% hydrogenated egg PC (EPCH). The polyunsaturated fatty acid content (C18:2+C18:3+C20:4) of soy, egg and hydrogenated PC was 70%, 20% and 0%, respectively. Total liver lipid was significantly lower in SPCH and EPCH vs. HF (8.7 ± 0.1 and 8.5 ± 0.5 vs. 11.8 ± 0.6g/100, P<0.05), but not in SPC or EPC. SPCH and EPCH had significantly lower levels of hepatic cholesterol (-52% and -53% vs. HF, respectively). Bioactive lipids (i.e., sphingomyelin and ceramide) were also lower in the liver of SPCH and EPCH rather than in SPC or EPC. Hepatic expression of genes controlling fatty acid synthesis and catabolism were not significantly affected by dietary PC. However, hepatic expression of HMGCR, LDLR and SREBP2 was higher and that of ABCA1, ABCG5 and ABCG8 was reduced in SPCH and EPCH vs. HF. These results demonstrate that hydrogenated PC supplementation reduces hepatic lipid levels in mice fed a high-fat diet supporting the concept that the ability of dietary PC to lower hepatic lipid levels is not due to its content of polyunsaturated fatty acids.