Natalie A. Mellett

Specific plasma lipid classes and phospholipid fatty acids indicative of dairy food consumption associate with insulin sensitivity

BACKGROUND Reports have suggested that the consumption of dairy foods may reduce risk of type 2 diabetes on the basis of evidence of raised circulating ruminant fatty acids. OBJECTIVE We determined whether certain phospholipid species and fatty acids that are associated with full-fat dairy consumption may also be linked to diminished insulin resistance. DESIGN Four variables of insulin resistance and sensitivity were defined from oral-glucose-tolerance tests in 86 overweight and obese subjects with metabolic syndrome. Plasma phospholipids, sphingolipids, and fatty acids were determined by using a lipidomic analysis and gas chromatography-mass spectrometry to provide objective markers of dairy consumption. Food records provided information on dairy products. Associations were determined by using linear regression analyses adjusted for potential confounders age, sex, systolic blood pressure, waist:hip ratio, or body mass index (BMI) and corrected for multiple comparisons. RESULTS Lysophosphatidylcholine, lyso-platelet-activating factor, and several phospholipid fatty acids correlated directly with the number of servings of full-fat dairy foods. Lysophosphatidylcholine and lyso-platelet-activating factor were also associated directly with insulin sensitivity when accounting for the waist:hip ratio (Matsuda index unadjusted, P < 0.001 for both; adjusted for multiple comparisons, P < 0.02 for both) and inversely with insulin resistance (fasting insulin unadjusted, P < 0.001 for both; adjusted, P = 0.04 and P < 0.05, respectively; homeostasis model assessment of insulin resistance adjusted, P = 0.04 for both; post-glucose insulin area under the plasma insulin curve during the 120 min of the test adjusted, P < 0.01 for both). The substitution of BMI for the waist:hip ratio attenuated associations modestly. Phospholipid fatty acid 17:0 also tended to be associated directly with insulin sensitivity and inversely with resistance. CONCLUSION Variables of insulin resistance were lower at higher concentrations of specific plasma phospholipids that were also indicators of full-fat dairy consumption. This trial was registered at clinicaltrials.gov as NCT00163943.

Effects of low-fat or full-fat fermented and non-fermented dairy foods on selected cardiovascular biomarkers in overweight adults

The association between consumption of full-fat dairy foods and CVD may depend partly on the nature of products and may not apply to low-fat dairy foods. Increased circulating levels of inflammatory biomarkers after consumption of dairy product-rich meals suggest an association with CVD. In the present study, we tested the effects of low-fat and full-fat dairy diets on biomarkers associated with inflammation, oxidative stress or atherogenesis and on plasma lipid classes. Within full-fat dairy diets, we also compared fermented v. non-fermented products. In a randomised cross-over study, twelve overweight/obese subjects consumed during two 3-week periods two full-fat dairy diets containing either yogurt plus cheese (fermented) or butter, cream and ice cream (non-fermented) or a low-fat milk plus yogurt diet, with the latter being consumed between and at the end of the full-fat dairy dietary periods. The concentrations of six inflammatory and two atherogenic biomarkers known to be raised in CVD were measured as well as those of plasma F2-isoprostanes and lipid classes. The concentrations of six of the eight biomarkers tended to be higher on consumption of the low-fat dairy diet than on that of the fermented dairy diet and the concentrations of two plasmalogen lipid classes reported to be associated with increased oxidisability were also higher on consumption of the low-fat dairy diet than on that of the fermented dairy diet (P< 0.001), although plasma F2-isoprostane concentrations did not differ on consumption of any of the diets. On the other hand, the concentrations of plasma sphingomyelin and IL-6 were significantly higher on consumption of the non-fermented dairy diet than on that of the low-fat dairy diet (P< 0.02). In conclusion, short-term diets containing low-fat dairy products did not lead to a more favourable biomarker profile associated with CVD risk compared with the full-fat dairy products, suggesting that full-fat fermented dairy products may be the more favourable.

Plasma Lipidomic Profiles Improve on Traditional Risk Factors for the Prediction of Cardiovascular Events in Type 2 Diabetes Mellitus

Background: Clinical lipid measurements do not show the full complexity of the altered lipid metabolism associated with diabetes mellitus or cardiovascular disease. Lipidomics enables the assessment of hundreds of lipid species as potential markers for disease risk. Methods: Plasma lipid species (310) were measured by a targeted lipidomic analysis with liquid chromatography electrospray ionization–tandem mass spectrometry on a case-cohort (n=3779) subset from the ADVANCE trial (Action in Diabetes and Vascular Disease: Preterax and Diamicron-MR Controlled Evaluation). The case-cohort was 61% male with a mean age of 67 years. All participants had type 2 diabetes mellitus with ≥1 additional cardiovascular risk factors, and 35% had a history of macrovascular disease. Weighted Cox regression was used to identify lipid species associated with future cardiovascular events (nonfatal myocardial infarction, nonfatal stroke, and cardiovascular death) and cardiovascular death during a 5-year follow-up period. Multivariable models combining traditional risk factors with lipid species were optimized with the Akaike information criteria. C statistics and NRIs were calculated within a 5-fold cross-validation framework. Results: Sphingolipids, phospholipids (including lyso- and ether- species), cholesteryl esters, and glycerolipids were associated with future cardiovascular events and cardiovascular death. The addition of 7 lipid species to a base model (14 traditional risk factors and medications) to predict cardiovascular events increased the C statistic from 0.680 (95% confidence interval [CI], 0.678–0.682) to 0.700 (95% CI, 0.698–0.702; P<0.0001) with a corresponding continuous NRI of 0.227 (95% CI, 0.219–0.235). The prediction of cardiovascular death was improved with the incorporation of 4 lipid species into the base model, showing an increase in the C statistic from 0.740 (95% CI, 0.738–0.742) to 0.760 (95% CI, 0.757–0.762; P<0.0001) and a continuous net reclassification index of 0.328 (95% CI, 0.317–0.339). The results were validated in a subcohort with type 2 diabetes mellitus (n=511) from the LIPID trial (Long-Term Intervention With Pravastatin in Ischemic Disease). Conclusions: The improvement in the prediction of cardiovascular events, above traditional risk factors, demonstrates the potential of plasma lipid species as biomarkers for cardiovascular risk stratification in diabetes mellitus. Clinical Trial Registration: URL: https://clinicaltrials.gov. Unique identifier: NCT00145925.

Zebrafish embryonic lipidomic analysis reveals that the yolk cell is metabolically active in processing lipid

The role of lipids in providing energy and structural cellular components during vertebrate development is poorly understood. To elucidate these roles further, we visualized lipid deposition and examined expression of key lipid-regulating genes during zebrafish embryogenesis. We also conducted a semiquantitative analysis of lipidomic composition using liquid chromatography (LC)-mass spectrometry. Finally, we analyzed processing of boron-dipyrromethene (BODIPY) lipid analogs injected into the yolk using thin layer chromatography. Our data reveal that the most abundant lipids in the embryo are cholesterol, phosphatidylcholine, and triglyceride. Moreover, we demonstrate that lipids are processed within the yolk prior to mobilization to the embryonic body. Our data identify a metabolically active yolk and body resulting in a dynamic lipid composition. This provides a foundation for studying lipid biology during normal or pharmacologically compromised embryogenesis.

Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites

C-reactive protein (CRP) concentrations rise in response to tissue injury or infection. Circulating pentameric CRP (pCRP) localizes to damaged tissue where it leads to complement activation and further tissue damage. In-depth knowledge of the pCRP activation mechanism is essential to develop therapeutic strategies to minimize tissue injury. Here we demonstrate that pCRP by binding to cell-derived microvesicles undergoes a structural change without disrupting the pentameric symmetry (pCRP*). pCRP* constitutes the major CRP species in human-inflamed tissue and allows binding of complement factor 1q (C1q) and activation of the classical complement pathway. pCRP*–microvesicle complexes lead to enhanced recruitment of leukocytes to inflamed tissue. A small-molecule inhibitor of pCRP (1,6-bis(phosphocholine)-hexane), which blocks the pCRP–microvesicle interactions, abrogates these proinflammatory effects. Reducing inflammation-mediated tissue injury by therapeutic inhibition might improve the outcome of myocardial infarction, stroke and other inflammatory conditions.

Postprandial Plasma Phospholipids in Men Are Influenced by the Source of Dietary Fat

BACKGROUND Postprandial lipemia represents a risk factor for chronic diseases, including type 2 diabetes. Little is known about the effect of dietary fat on the plasma lipidome in the postprandial period. OBJECTIVE The objective of this study was to assess the effect of dairy fat and soy oil on circulating postprandial lipids in men. METHODS Men (40-60 y old, nonsmokers; n = 16) were randomly assigned in a crossover design to consume 2 breakfast meals of dairy-based or soy oil-based foods. The changes in the plasma lipidome during the 4-h postprandial period were analyzed with electrospray ionization tandem mass spectrometry and included 316 lipid species in 23 classes and subclasses, representing sphingolipids, phospholipids, glycerolipids, and sterols. RESULTS Nonparametric Friedman tests showed significant changes in multiple plasma lipid classes, subclasses, and species in the postprandial period after both dairy and soy meals. No difference was found in triglyceridemia after each meal. However, 6 endogenous lipid classes increased after dairy but decreased after soy (P < 0.05), including ether-linked phospholipids and plasmalogens and sphingomyelin (not present in soy), dihexosylceramide, and GM3 ganglioside. Phosphatidylcholine and phosphatidylinositol were not affected by the soy meal but were significantly elevated after the dairy meal (8.3% and 16%, respectively; P < 0.05). CONCLUSIONS The changes in postprandial plasma phospholipids in men relate to the diet composition and the relative size of the endogenous phospholipid pools. Despite similar lipemic responses as measured by changes in triglyceride concentrations, the differential responses to dairy and soy meals derived through lipidomic analysis of phospholipids suggest differences in the metabolism of soybean oil and dairy fat. The increased concentrations of plasmalogens, with potential antioxidant capacity, in the postprandial period after dairy but not soy meals may represent a further important difference in the response to these sources of fat. The trial was registered at www.anzctr.org.au as ACTRN12610000562077.

Fetal inhibition of inflammation improves disease phenotypes in harlequin ichthyosis

Harlequin ichthyosis (HI) is a severe skin disease which leads to neonatal death in ∼50% of cases. It is the result of mutations in ABCA12, a protein that transports lipids required to establish the protective skin barrier needed after birth. To better understand the life-threatening newborn HI phenotype, we analysed the developing epidermis for consequences of lipid dysregulation in mouse models. We observed a pro-inflammatory signature which was characterized by chemokine upregulation in embryonic skin which is distinct from that seen in other types of ichthyosis. Inflammation also persisted in grafted HI skin. To examine the contribution of inflammation to disease development, we overexpressed interleukin-37b to globally suppress fetal inflammation, observing considerable improvements in keratinocyte differentiation. These studies highlight inflammation as an unexpected contributor to HI disease development in utero, and suggest that inhibiting inflammation may reduce disease severity.

The effects of long-term saturated fat enriched diets on the brain lipidome

The brain is highly enriched in lipids, where they influence neurotransmission, synaptic plasticity and inflammation. Non-pathological modulation of the brain lipidome has not been previously reported and few studies have investigated the interplay between plasma lipid homeostasis relative to cerebral lipids. This study explored whether changes in plasma lipids induced by chronic consumption of a well-tolerated diet enriched in saturated fatty acids (SFA) was associated with parallel changes in cerebral lipid homeostasis. Male C57Bl/6 mice were fed regular chow or the SFA diet for six months. Plasma, hippocampus (HPF) and cerebral cortex (CTX) lipids were analysed by LC-ESI-MS/MS. A total of 348 lipid species were determined, comprising 25 lipid classes. The general abundance of HPF and CTX lipids was comparable in SFA fed mice versus controls, despite substantial differences in plasma lipid-class abundance. However, significant differences in 50 specific lipid species were identified as a consequence of SFA treatment, restricted to phosphatidylcholine (PC), phosphatidylethanolamine (PE), alkyl-PC, alkenyl-PC, alkyl-PE, alkenyl-PE, cholesterol ester (CE), diacylglycerol (DG), phosphatidylinositol (PI) and phosphatidylserine (PS) classes. Partial least squares regression of the HPF/CTX lipidome versus plasma lipidome revealed the plasma lipidome could account for a substantial proportion of variation. The findings demonstrate that cerebral abundance of specific lipid species is strongly associated with plasma lipid homeostasis.

Breaking Up Prolonged Sitting Alters the Postprandial Plasma Lipidomic Profile of Adults With Type 2 Diabetes

Context Postprandial dysmetabolism in type 2 diabetes (T2D) is exacerbated by prolonged sitting and may trigger inflammation and oxidative stress. It is unknown what impact countermeasures to prolonged sitting have on the postprandial lipidome. Objective In this study, we investigated the effects of regular interruptions to sitting, compared with prolonged sitting, on the postprandial plasma lipidome. Design Randomized crossover experimental trial. Setting Participants underwent three 7-hour conditions: uninterrupted sitting (SIT); light-intensity walking interruptions (LW); and simple resistance activity interruptions (SRA). Participants and Samples Baseline (fasting) and 7-hour (postprandial) plasma samples from 21 inactive overweight/obese adults with T2D were analyzed for 338 lipid species using mass spectrometry. Main Outcome Measures Using mixed model analysis (controlling for baseline outcome variable, gender, body mass index, and condition order), the percentage change in lipid species (baseline to 7 hours) was compared between conditions with Benjamini-Hochberg correction. Results Thirty-seven lipids were different between conditions (P < 0.05). Compared with SIT, postprandial elevations in diacylglycerols, triacylglycerols, and phosphatidylethanolamines were attenuated in LW and SRA. Plasmalogens and lysoalkylphosphatidylcholines were reduced in SIT, compared with attenuated reductions or elevations in LW and SRA. Phosphatidylserines were elevated with LW, compared with reductions in SIT and SRA. Conclusion Compared with SIT, LW and SRA were associated with reductions in lipids associated with inflammation; increased concentrations of lipids associated with antioxidant capacity; and differential changes in species associated with platelet activation. Acutely interrupting prolonged sitting time may impart beneficial effects on the postprandial plasma lipidome of adults with T2D. Evidence on longer-term intervention is needed.

A comprehensive lipidomic screen of pancreatic β-cells using mass spectroscopy defines novel features of glucose-stimulated turnover of neutral lipids, sphingolipids and plasmalogens

Objective Glucose promotes lipid remodelling in pancreatic β-cells, and this is thought to contribute to the regulation of insulin secretion, but the metabolic pathways and potential signalling intermediates have not been fully elaborated. Methods Using mass spectrometry (MS) we quantified changes in approximately 300 lipid metabolites in MIN6 β-cells and isolated mouse islets following 1 h stimulation with glucose. Flux through sphingolipid pathways was also assessed in 3H-sphinganine-labelled cells using TLC. Results Glucose specifically activates the conversion of triacylglycerol (TAG) to diacylglycerol (DAG). This leads indirectly to the formation of 18:1 monoacylglycerol (MAG), via degradation of saturated/monounsaturated DAG species, such as 16:0_18:1 DAG, which are the most abundant, immediate products of glucose-stimulated TAG hydrolysis. However, 16:0-containing, di-saturated DAG species are a better direct marker of TAG hydrolysis since, unlike the 18:1-containing DAGs, they are predominately formed via this route. Using multiple reaction monitoring, we confirmed that in islets under basal conditions, 18:1 MAG is the most abundant species. We further demonstrated a novel site of glucose to enhance the conversion of ceramide to sphingomyelin (SM) and galactosylceramide (GalCer). Flux and product:precursor analyses suggest regulation of the enzyme SM synthase, which would constitute a separate mechanism for localized generation of DAG in response to glucose. Phosphatidylcholine (PC) plasmalogen (P) species, specifically those containing 20:4, 22:5 and 22:6 side chains, were also diminished in the presence of glucose, whereas the more abundant phosphatidylethanolamine plasmalogens were unchanged. Conclusion Our results highlight 18:1 MAG, GalCer, PC(P) and DAG/SM as potential contributors to metabolic stimulus-secretion coupling.