Dr Barbara Fielding

Postprandial TAG is lower in Black African-Caribbean men than in White European men, and this is likely driven by lower meal-derived VLDL-TAG in Black African-Caribbean men. This observation could suggest that fatty acid trafficking may be a less important determinant of cardiometabolic risk in Black Africans than in White European men. Black African-Caribbean (BAC) populations are at greater risk of cardiometabolic disease than White Europeans (WE), despite exhibiting lower fasting triacylglycerol (TAG) concentrations. However, limited data exist regarding postprandial fatty acid metabolism in BAC populations. This study determined the ethnic differences in postprandial fatty acid metabolism between overweight and obese WE and BAC men. WE [ n = 10, age 33.3 ± 1.7 yr; body mass index (BMI) = 26.8 (25.8–31.0) kg/m 2 ] and BAC [ n = 9, age 27.9 ± 1.0 yr; BMI = 27.5 (26.0–28.6) kg/m 2 ] men consumed two consecutive (at 0 and 300 min) moderate-to-high-fat meals—the first labeled with [U- 13 C]palmitate. The plasma concentration and appearance of meal-derived fatty acids in very-low-density lipoprotein (VLDL)-TAG, chylomicron-TAG, and nonesterified fatty acid (NEFA) were determined over an 8-h postprandial period. Indirect calorimetry with 13 CO 2 enrichment determined total and meal-derived fatty acid oxidation rates, and plasma β-hydroxybutyrate (3-OHB) concentration was measured to assess ketogenesis. BAC exhibited lower postprandial TAG [area under the curve (AUC 0–480 ) = 671 (563–802) vs. 469 (354–623) mmol/L/min, P = 0.022] and VLDL-TAG [AUC 0–480 = 288 ± 30 vs. 145 ± 27 mmol/L/min, P = 0.003] concentrations than WE. The appearance of meal-derived fatty acids in VLDL-TAG was lower in BAC than in WE (AUC 0–480 = 133 ± 12 vs. 78 ± 13 mmol/L/min, P = 0.007). Following the second meal, BAC showed a trend for lower chylomicron-TAG concentration [AUC 300–480 = 69 (51–93) vs. 43 (28–67) mmol/L/min, P = 0.057]. There were no ethnic differences in the appearance of chylomicron-TAG, cumulative fatty acid oxidation, and the NEFA:3-OHB ratio ( P > 0.05). In conclusion, BAC exhibit lower postprandial TAG concentrations compared with WE men, driven by lower VLDL-TAG concentrations and possibly lower chylomicron-TAG in the late postprandial period. These findings suggest that postprandial fatty acid trafficking may be a less important determinant of cardiometabolic risk in BAC than in WE men. NEW & NOTEWORTHY Postprandial TAG is lower in Black African-Caribbean men than in White European men, and this is likely driven by lower meal-derived VLDL-TAG in Black African-Caribbean men. This observation could suggest that fatty acid trafficking may be a less important determinant of cardiometabolic risk in Black Africans than in White European men.

Background: Interesterified (IE) fats are widely used in place of trans fats; however, little is known about their metabolism. Objective: To test the impact of a commonly consumed IE versus a non-IE equivalent fat on in vivo postprandial and in vitro lipid metabolism, compared with a reference oil (rapeseed oil; RO). Design: A double-blinded, 3-phase crossover, randomized controlled trial was performed in healthy adults (n=20) aged 45-75 years. Postprandial plasma triacylglycerol (TG) and lipoprotein responses (including stable isotope tracing) to a test meal (50g fat) were evaluated over 8 hours. The test fats were IE 80:20 palm stearin/palm kernel fat, an identical non-IE fat, and RO (control). In vitro, mechanisms of digestion were explored using a dynamic gastric model (DGM). Results: Plasma TG 8h incremental area under the curves were lower following non-IE versus RO (-1.7 mmol/L.h (95% confidence interval -3.3, -0.0)), but there were no differences between IE and RO nor IE and non-IE. Low density lipoprotein (LDL) particles were smaller following IE and non-IE versus RO (P=0.005). Extra, extra large (XXL)-, extra large (XL)- and large (L)- VLDL particle concentrations were higher following IE and non-IE versus RO at 6-8 h (P

Experimental inversion of circadian and behavioural rhythms by 12-hours adversely affects markers of metabolic health. We investigated the effects of a more modest 5-hour delay in behavioural cycles. Fourteen participants completed an 8-day in-patient laboratory protocol, with controlled sleep-wake opportunities, light-dark cycles, and diet. The 5-hour delay in behavioural cycles was induced by delaying sleep opportunity. We measured: melatonin to confirm central circadian phase; fasting markers and postprandial metabolism; energy expenditure; subjective sleepiness; and appetite, throughout the waking period. After the phase delay, there was slower gastric emptying at breakfast, lower fasting plasma glucose, higher postprandial plasma glucose and triglycerides, and lower thermic effect of feeding. Any changes were abolished or attenuated within 48-72 hours. These data extend our previous findings, which showed no time-of-day effect in healthy adults on daytime energy expenditure or thermic effect of feeding when accounting for circadian variation in resting metabolic rate.

Human studies suggest that reducing dietary saturated fatty acids (SFAs) via replacing it with unsaturated fatty acids (UFAs) has a beneficial effect on fasting low-density lipoprotein cholesterol (LDL-C)(Reference Siri-Tarino, Sun and Hu1). However, significant variation in LDL-C concentration has been observed in response to change in dietary SFA intake(Reference Vafeiadou, Weech and Altowaijri2,Reference Jebb, Lovegrove and Griffin3) and there is considerable interest in potential determinants underlying the responsiveness of LDL-C to fat intake. Dietary fat composition affects body fat distribution with higher SFA intakes associated with abdominal obesity and greater cardiovascular disease (CVD) risk(Reference Rosqvist, Iggman and Kullberg4). However, it is not clear whether the variability in LDL-C response to dietary SFA is related to changes in body composition.

Objective This work aimed to investigate the effect of the SGLT2 inhibitor, dapagliflozin (DAPA), on cardiac function and the metabolic and hormonal response to moderate exercise in people with type 2 diabetes. Methods This was a double-blind, placebo-controlled crossover study with a 4-week washout period. Nine participants were randomly assigned to receive either 4 weeks of DAPA or 4 weeks of placebo. After each treatment, they underwent an exercise protocol with 2 consecutive 10-minute stages at a constant load corresponding to 40% and 70% maximal oxygen consumption (VO2max), coupled with hormonal and metabolic analysis. A blinded transthoracic echocardiogram was performed 3 days later. Results During the exercise protocol, glucose and lactate were lower (P < .0001 and P < .05, respectively) and β-hydroxybutyrate (BOBH) and growth hormone (GH) were higher (P < .0005 and P = .01) following DAPA treatment compared to placebo. There was a trend for lower insulin with DAPA. Adrenalin, noradrenalin, and glucagon were not different. Following DAPA participants demonstrated an increased mean peak diastolic mitral annular velocity (e’) in comparison to placebo (P = .03). The indexed left atrial volume and right ventricular e” were reduced following DAPA compared with placebo (P = .045 and P = .042, respectively). Arterial stiffness was not different between treatments (DAPA 9.35 ± 0.60 m/s; placebo 9.07 ± 0.72 m/s). Conclusion During exercise, GH may be more important than catecholamines in driving the shift from glucose to fatty acid metabolism by SGLT2 inhibitors. The 4-week crossover design showed changes in cardiac function were rapid in onset and reversible.

Using the power of stable isotope techniques, our study explored the physiological effects of the SGLT2 inhibitor dapagliflozin on glucose flux, lipolysis, and ketone body concentration in people with acute absolute insulin withdrawal (1). The power of our study from the clinical perspective was the crossover design with each individual undergoing an identical insulin withdrawal protocol with the only difference being the presence or absence of an SGLT2 inhibitor.

mpaired regulation of immune function characterised by chronic inflammation together with a declining protective immune response is a major challenge to healthy ageing. It is therefore important to understand the mechanisms that regulate immune function and the impact of ageing upon such processes. Appropriate induction and resolution of the immune response requires adequate availability of polyunsaturated fatty acids (PUFAs) for incorporation into cell membranes. However, humans are unable to synthesise PUFAs de novo and are dependent upon dietary intake for pre-formed PUFAs or synthesis by the liver from the essential fatty acids, linoleic acid (LA, 18:2n-6) and alpha-linolenic acid (aLNA, 18:3n-3). We have shown that activation of peripheral blood mononuclear cells (PBMCs) 37 increases PUFA biosynthesis from essential fatty acids via a mechanism that involves altered epigenetic regulation of a key gene in the pathway. Moreover, induction of PUFA synthesis is directly involved in the regulation of lymphocyte activation and proliferation. The aim of the BBSRC responsive mode award ‘How does polyunsaturated fatty acid biosynthesis regulate T lymphocyte function?’ is to determine how PUFA biosynthesis regulates T cell function and the effect of ageing on this process. The project will identify points of regulation in the biosynthetic pathway and how these might influence the capacity for up-regulation of PUFA synthesis in older individuals. We will use stable isotope tracers of LA and aLNA to determine whether newly synthesised PUFAs are preferential substrates for synthesis of lipid mediators and whether they are involved in formation of membrane microdomains that mediate cell signalling.

Immune function changes across the life stages; for example, senior adults exhibit a tendency towards a weaker cell-mediated immune response and a stronger inflammatory response than younger adults. This might be partly mediated by changes in oxylipin synthesis across the life course. Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that modulate immune function and inflammation. A number of PUFAs are precursors to oxylipins, including the essential fatty acids (EFAs) linoleic acid (LA) and α-linolenic acid (ALA). LA and ALA are also substrates for synthesis of longer chain PUFAs. Studies with stable isotopes have shown that the relative amounts of LA and ALA can influence their partitioning by T lymphocytes between conversion to longer chain PUFAs and to oxylipins. It is not known whether the relative availability of EFA substrates influences the overall pattern of oxylipin secretion by human T cells or if this changes across the life stages. To address this, the oxylipin profile was determined in supernatants from resting and mitogen activated human CD3+ T cell cultures incubated in medium containing an EFA ratio of either 5:1 or 8:1 (LA : ALA). Furthermore, oxylipin profiles in supernatants of T cells from three life stages, namely fetal (derived from umbilical cord blood), adults and seniors, treated with the 5:1 EFA ratio were determined. The extracellular oxylipin profiles were affected more by the EFA ratio than mitogen stimulation such that n-3 PUFA-derived oxylipin concentrations were higher with the 5:1 EFA ratio than the 8:1 ratio, possibly due to PUFA precursor competition for lipoxygenases. 47 oxylipin species were measured in all cell culture supernatants. Extracellular oxylipin concentrations were generally higher for fetal T cells than for T cells from adult and senior donors, although the composition of oxylipins was similar across the life stages. The contribution of oxylipins towards an immunological phenotype might be due to the capacity of T cells to synthesize oxylipins rather than the nature of the oxylipins produced.

A Nutrition Society member-led meeting was held on 9 January 2020 at The University of Surrey, UK. Sixty people registered for the event, and all were invited to participate, either through chairing a session, presenting a ‘3 min lightning talk’ or by presenting a poster. The meeting consisted of an introduction to the topic by Dr Barbara Fielding, with presentations from eight invited speakers. There were also eight lightning talks and a poster session. The meeting aimed to highlight recent research that has used stable isotope tracer techniques to understand human metabolism. Such studies have irrefutably shaped our current understanding of metabolism and yet remain a mystery to many. The meeting aimed to de-mystify their use in nutrition research.

A high fructose intake exacerbates postprandial plasma triacylglycerol (TAG) concentration, an independent risk factor for cardiovascular disease, although it is unclear whether this is due to increased production or impaired clearance of triacylglycerol (TAG)-rich lipoproteins. We determined the in vivo acute effect of fructose on postprandial intestinal and hepatic lipoprotein TAG kinetics and de novo lipogenesis (DNL). Five overweight men were studied twice, 4 weeks apart. They consumed hourly mixed-nutrient drinks that were high-fructose (30% energy) or low-fructose (

Delayed wound healing is a devastating complication of diabetes and supplementation with fish oil, a source of anti-inflammatory omega-3 (ω-3) fatty acids including eicosapentaenoic acid (EPA), seems an appealing treatment strategy. However, some studies have shown that ω-3 fatty acids may have a deleterious effect on skin repair and the effects of oral administration of EPA on wound healing in diabetes are unclear. We used streptozotocin-induced diabetes as a mouse model to investigate the effects of oral administration of an EPA-rich oil on wound closure and quality of new tissue formed. Gas chromatography analysis of serum and skin showed that EPA-rich oil increased the incorporation of ω-3 and decreased ω-6 fatty acids, resulting in reduction of the ω-6/ω-3 ratio. On the tenth day after wounding, EPA increased production of IL-10 by neutrophils in the wound, reduced collagen deposition, and ultimately delayed wound closure and impaired quality of the healed tissue. This effect was PPAR-γ-dependent. EPA and IL-10 reduced collagen production by fibroblasts in vitro. In vivo, topical PPAR-γ-blockade reversed the deleterious effects of EPA on wound closure and on collagen organization in diabetic mice. We also observed a reduction in IL-10 production by neutrophils in diabetic mice treated topically with the PPAR-γ blocker. These results show that oral supplementation with EPA-rich oil impairs skin wound healing in diabetes, acting on inflammatory and non-inflammatory cells.

The essential fatty acid (EFA) α-linolenic acid (ALA, 18:3n-3) can be metabolised into longer chain n-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid and docosahexaenoic acid. ALA can also be oxidised into immunomodulatory lipid mediators, such as 18-carbon oxylipins, including hydroxyoctadecatrienoic acids (HOTrEs). Because HOTrE synthesis is impaired in inflammatory disease, we hypothesised that partitioning of ALA towards HOTrE synthesis is reduced in T cells from older individuals as a possible mechanism in age-related immune dysregulation known as immunosenescence(Reference Rodriguez, Lalinde Ruiz and Llano León1).To test this, peripheral blood CD3+ T cells from healthy younger adult volunteers (18–30 years; n = 10) and older adult volunteers (58–74 years; n = 6) were cultured for 48 h, with or without concanavalin A (10 μg/mL) in 10% (v/v) pooled donor plasma, in media with a 5:1 linoleic acid (LA, 18:2 n-6) to ALA ratio. Total ALA included [13C] ALA, to trace the relative partitioning of ALA. ALA metabolites were detected either by gas chromatography-mass spectrometry for cellular PUFA or by LC-MS/MS for oxylipins in cell culture supernatant (Reference Von Gerichten, West and Irvine2). Metabolite to ALA ratios were calculated and for the primary PUFA synthesised from ALA in T cells, eicosatrienoic acid ([13C]20:3n-3), and the most abundantly oxidised metabolite of ALA, [13C]9-HOTrE. Multiple t-test (unpaired, two-tailed) with Holm-Sidak correction was performed for statistical analysis on log transformed data (GraphPad Prism 8.4.3). Results for stimulated and unstimulated cells were similar; only results for stimulated cells are reported here. Oxylipin synthesis, measured as [13C]9-HOTrE / ALA was lower in cells from older adults compared to younger adults (median (range), 2.7 (1.1–4.0) vs 5.4 (2.1–9.6), P = 0.01). We then compared the relative partitioning of ALA into the alternative metabolic pathway and found that [13C]9-HOTrE / ALA was markedly higher than [13C]20:3n-3 / ALA (5.4 (2.1–9.6) vs 0.02 (0.004–0.036) , P < 0.001), in younger as well as in older adults (2.7 (1.1–4.0) vs 0.04 (0.02–0.08), P < 0.001).In conclusion, using a stable isotope tracer, we found that mitogen- stimulated T cells took up ALA added to the culture medium and preferentially used it for the constitutive production of 9-HOTrE, rather than synthesis of longer chain PUFAs in both younger and older adults. However, partitioning altered with age, towards 20:3n-3 synthesis rather than 18-carbon oxylipin production, which is consistent with induction of a more inflammatory phenotype in older individuals. This has implications for understanding the role of essential fatty acids in immunosenescence.

Longer chain polyunsaturated fatty acids (LCPUFA) ≥ 20 carbons long are required for leukocyte function. These can be obtained from the diet, but there is some evidence that leukocytes can convert essential fatty acids (EFA) into LCPUFA. We used stable isotope tracers to investigate LCPUFA biosynthesis, and the effect of different EFA substrate ratios, in human T lymphocytes. CD3+ T cells were incubated for up to 48 hours with or without concanavalin A in media containing a 18:2n-6 : 18:3n-3 (EFA) ratio of either 5:1 or 8:1, and [13C]18:3n-3 plus [d5]18:2n-6. Mitogen stimulation increased the amounts of 16:1n 7, 18:1n 9, 18:2n 6, 20:3n 6, 20:4n 6, 18:3n 3 and 20:5n 3 in T cells. Expression of the activation marker CD69 preceded increased FADS2 and FADS1 mRNA expression, and increased amounts of [d5]20:2n 6 and [13C]20:3n 3 at 48 hours. 22 carbon n 6 or n 3 LCPUFA synthesis was not detected, consistent with the absence of ELOVL2 expression. An EFA ratio of 8:1 reduced 18:3n 3 conversion and enhanced 20:2n 6 synthesis compared to a 5:1 ratio. [d5]9- and [d5]-13-hydroxyoctadecadienoic (HODE) and [13C]9- and [13C]13-hydroxyoctadecatrienoic acids (HOTrE) were the major labelled oxylipins in culture supernatants; labelled oxylipins ≥ 20 carbons were not detected. An EFA ratio of 8:1 suppressed 9- and 13-HOTrE synthesis, but there was no significant effect on 9- and 13-HODE synthesis. These findings suggest that partitioning of newly assimilated EFA between LCPUFA synthesis and hydroxyoctadecaenoic acid may be a metabolic branch point in T cell EFA metabolism that has implications for understanding the effects of dietary fats on T lymphocyte function.

Objective: To determine the effect of SGLT2 inhibitor dapagliflozin on glucose flux, lipolysis and ketone body concentrations during insulin withdrawal in people with type 1 diabetes. Research Design and Methods: A double-blind placebo controlled crossover study with a 4-week wash out period was performed in 12 people with type 1 diabetes using insulin pump therapy. Participants received dapagliflozin or placebo in random order for 7 days. Stable isotopes were infused to measure the rate of glucose production (Ra), disappearance (Rd) and lipolysis. At isotopic steady state insulin was withdrawn and the study terminated after 600 minutes or earlier if blood glucose reached 18mmol/L, bicarbonate 27 and

Objectives: Experimental inversion of circadian and behavioural rhythms by 12-hours adversely effects markers of metabolic health. The objective of this work was to investigate the effects of a more modest 5-hour delay in behavioural cycles (as observed in e.g. trans-Atlantic or trans-continental jetlag) on circadian and metabolic markers. Methods: Fourteen participants completed an 8-day inpatient laboratory protocol, with controlled sleep-wake opportunities, light-dark cycles, meal timing and diet composition. The 5-hour delay in sleep/wake and mealtimes was induced by delaying sleep opportunity. We measured melatonin to assess central circadian phase; markers of postprandial metabolism; subjective sleepiness, and appetite. Results: Melatonin rhythms gradually adapted to the new behavioural cycle. In the 4-hours after the phase delay, there was slower gastric emptying at breakfast, lower fasting plasma glucose, higher postprandial plasma glucose and triglycerides, and lower thermic effect of feeding (p < 0.05). These changes were abolished or attenuated within 48–72 hours of the phase delay. Conclusions: Moderate (5-hour) circadian desynchrony only transiently disrupts metabolism. Occasional jetlag may therefore pose low metabolic health risk

Increased postprandial glycaemia and reduced insulin sensitivity are associated with development of Type 2 Diabetes (T2D). Maintaining a normal glucose response is important both for healthy individuals, for disease prevention, and for those with T2D, to prevent development of diabetes-related complications. Chilling previously-cooked starchy carbohydrate (CHO) results in retrogradation of some of the starch to form resistant starch (RS). RS is not absorbed in the small intestine and consequently does not contribute to the postprandial glucose excursion. Reheating the CHO, however, reverses this process, reducing some of the RS content. RS type 5 is formed in the laboratory by heating starch with free fatty acids; there is limited evidence for its formation using domestic cooking methods and real foods. Furthermore it is unclear whether this would translate into a noticeable effect on postprandial glucose metabolism. In this randomised crossover study, 8 participants attended two study days; at one they consumed a freshly cooked mashed potato meal (203 g boiled potato, 25 g butter) at the other they consumed an identical meal which had been chilled for 66 h then microwave reheated. The potatoes were intrinsically labelled with [U-13C]starch, and participants received a variable [6,6-2H2]glucose infusion, allowing detailed glucose flux modelling. Venous blood samples were taken for 6 h postprandially. There was no significant effect on postprandial glucose, however repeated measures ANOVA on postprandial insulin time-point data found a significant difference between meals (p = 0.026), with a 24% reduction in incremental area under the curve (0–120 min) and 21% reduction in insulin peak between freshly cooked and reheated meals. There were no significant effects on rate of appearance of glucose into the plasma from the gut (Ra) or on endogenous (hepatic) glucose production (EGP), however there was a strong trend for a reduced rate of glucose disposal (uptake into tissues, Rd) following the reheated meal (p = 0.054). It is hypothesised that RS was formed in the reheated meal by the chilling and reheating process; this will be verified by in vitro work later in the project. The attenuation of the postprandial insulin response with no significant effect on EGP suggests enhanced hepatic insulin sensitivity following the reheated meal as a possible mechanism for the effects of RS on postprandial glycaemia. This study demonstrates that making simple changes to the way a starchy carbohydrate meal is prepared can have significant beneficial effects on postprandial glucose metabolism.

Rationale for the dietary guideline to replace saturated (SFA) with unsaturated fatty acids (UFA) to reduce risk of atherosclerotic cardiovascular disease (ASCVD) rests heavily on the lowering of serum low density lipoprotein cholesterol (LDL-C)(Reference Mensink1). However, there is evidence to suggest that this dietary exchange may be associated with favourable effects on other cardiometabolic risk factors, including non-high-density lipoprotein-cholesterol (non-HDL-C), a measure of all atherogenic lipoproteins in serum(Reference Pinart, Jeran and Boeing2). The aim of this study was to determine the effects of dietary SFA on serum HDL-C, non-HDL-C, and remnant-cholesterol in the RISSCI-1 study.

OBJECTIVE To determine the effect of the sodium–glucose cotransporter 2 inhibitor dapagliflozin on glucose flux, lipolysis, and ketone body concentrations during insulin withdrawal in people with type 1 diabetes. RESEARCH DESIGN AND METHODS A double-blind, placebo-controlled crossover study with a 4-week washout period was performed in 12 people with type 1 diabetes using insulin pump therapy. Participants received dapagliflozin or placebo in random order for 7 days. Stable isotopes were infused to measure the glucose Ra, Rd, and lipolysis. At isotopic steady state, insulin was withdrawn, and the study was terminated after 600 min or earlier if blood glucose reached 18 mmol/L, bicarbonate 27 and

Perioperative nutrition aims to replenish nutritional stores before surgery and reduce postoperative complications. 'Immunonutrition' (including omega-3 fatty acids) may modulate the immune system and attenuate the postoperative inflammatory response. Hitherto, immunonutrition has overwhelmingly been administered in the postoperative period-however, this may be too late to provide benefit. A systematic literature search using MEDLINE and EMBASE for randomized controlled trials (RCTs). Perioperative major gastrointestinal surgery. Patients undergoing major gastrointestinal surgery. Omega-3 fatty acid supplementation commenced in the preoperative period, with or without continuation into postoperative period. The effect of preoperative omega-3 fatty acids on inflammatory response and clinical outcomes. 833 studies were identified. After applying inclusion and exclusion criteria, 12 RCTs, involving 1456 randomized patients, were included. Ten articles exclusively enrolled patients with cancer. Seven studies used a combination of EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) as the intervention and five studies used EPA alone. Eight out of 12 studies continued preoperative nutritional support into the postoperative period.Of the nine studies reporting mortality, no difference was seen. Duration of hospitalisation ranged from 4.5 to 18 days with intervention and 3.5 to 23.5 days with control. Omega-3 fatty acids had no effect on postoperative C-reactive protein and the effect on cytokines (including tumor necrosis factor-α, interleukin (IL)-6 and IL-10) was inconsistent. Ten of the 12 studies had low risk of bias, with one study having moderate bias from allocation and blinding. There is insufficient evidence to support routine preoperative omega-3 fatty acid supplementation for major gastrointestinal surgery, even when this is continued after surgery.

Introduction: Immune function changes across the life course; the fetal immune system is characterised by tolerance while that of seniors is less able to respond effectively to antigens and is more pro-inflammatory than in younger adults. Lipids are involved centrally in immune function but there is limited information about how T cell lipid metabolism changes during the life course. Methods and Results: We investigated whether life stage alters fatty acid composition, lipid droplet content and α-linolenic acid (18:3ω-3) metabolism in human fetal CD3⁺ T lymphocytes and in CD3⁺ T lymphocytes from adults (median 41 years) and seniors (median 70 years). Quiescent fetal T cells had higher saturated (SFA), monounsaturated fatty acid (MUFA), and ω-6 polyunsaturated fatty acid (PUFA) contents than adults or seniors. Activation-induced changes in fatty acid composition differed between life stages. The principal metabolic fates of [¹³C]18:3ω-3 were constitutive hydroxyoctadecatrienoic acid synthesis and β-oxidation and carbon recycling into SFA and MUFA. These processes declined progressively across the life course. Longer chain ω-3 PUFA synthesis was a relatively minor metabolic fate of 18:3ω-3 at all life stages. Fetal and adult T lymphocytes had similar lipid droplet contents, which were lower than in T cells from seniors. Variation in the lipid droplet content of adult T cells accounted for 62% of the variation in mitogen-induced CD69 expression, but there was no significant relationship in fetal cells or lymphocytes from seniors. Discussion: Together these findings show that fatty acid metabolism in human T lymphocytes changes across the life course in a manner that may facilitate the adaptation of immune function to different life stages.

Abstract: Dietary fructose has been linked to an increased post-prandial triglyceride (TG) level, which is an established independent risk factor for cardiovascular disease. Although much research has focused on the effects of fructose consumption on liver-derived very-low density lipoprotein (VLDL), emerging evidence also suggests that fructose may raise post-prandial TG levels by affecting the metabolism of enterocytes of the small intestine. Enterocytes have become well recognised for their ability to transiently store lipids following a meal and to thus control post-prandial TG levels according to the rate of chylomicron (CM) lipoprotein synthesis and secretion. The influence of fructose consumption on several aspects of enterocyte lipid metabolism are discussed, including de novo lipogenesis, apolipoprotein B48 and CM-TG production, based on the findings of animal and human isotopic tracer studies. Methodological issues affecting the interpretation of fructose studies conducted to date are highlighted, including the accurate separation of CM and VLDL. Although the available evidence to date is limited, disruption of enterocyte lipid metabolism may make a meaningful contribution to the hypertriglyceridaemia often associated with fructose consumption.

This pilot study investigated the effects of chilling and reheating a pasta-based meal on the postprandial glycaemic response. In this single-blind crossover study, 10 healthy volunteers consumed identical pasta meals (pasta, olive oil and tomato sauce), served either freshly prepared, chilled or chilled/reheated, on three separate randomised occasions. Capillary blood samples were taken for two hours postprandially. A significant difference in glucose Incremental Area Under the Curve (IAUC) was observed (p = 0.006), with the greatest difference observed between the freshly cooked and chilled/reheated meals (p = 0.041). Significant differences in incremental peak glucose were also observed (p = 0.018). These results suggest that making simple changes to domestic food processing methods can reduce the glycaemic excursion following a pasta meal, with the potential for health benefit.

With an increasing prevalence of diabetes worldwide, effective dietary strategies for blood glucose control are crucial. As carbohydrates make up approximately 50% of the diet, it is neither practical nor advisable to avoid them altogether. Most of the carbohydrate in the diet is derived from starch, found in potatoes, pasta, rice and bread. These foods are often processed in some way before consumption, yet little is known about the effects processing, such as chilling and reheating, has on the glycaemic response, particularly when the food is consumed in the context of a mixed meal. This article introduces the project, a BBSRC DRINC-funded initiative. Taking the potato as the model carbohydrate, this project will investigate, via and studies, the effects of domestic food processing techniques on the glycaemic response. A final study, utilising intrinsically labelled potato and a dual stable isotope methodology, will model glucose flux data to determine the underlying mechanisms of action.

We have previously shown that replacement of dietary saturated fat (SFA) with unsaturated fat (UFA) lowers serum LDL-cholesterol and increases the gene expression of LDL-receptors (LDL-R) in peripheral blood mononuclear cells, in the RISSCI-1 (Reading Imperial Surrey Saturated fat Cholesterol Intervention) study(Reference Ozen, Koutsos and Antoni1). This dietary-induced upregulation of LDL-R gene expression has been reported to occur in response to a reduction in endogenous cholesterol synthesis and intra- cellular free cholesterol. The latter, in part, is regulated by a reciprocal ‘push-pull’ relationship between the endogenous synthesis and intestinal absorption of cholesterol(Reference Alphonse and Jones2). The aim of the present investigation was to evaluate the effects of exchanging SFA for UFA on markers of intestinal absorption and endogenous synthesis of cholesterol.RISSCI-1 was a non-randomised, sequential dietary intervention study, in which 109 healthy men (age 48, SD 11y, BMI 25.1 kg/m2 SD 3.3) followed two iso-energetic diets; a high SFA (18% total energy (TE)) - lower UFA (15% TE) diet, followed by a lower SFA (10% TE) - high UFA (24% TE) diet, for 4 weeks each. Plasma non- cholesterol sterols, standardised for total cholesterol and expressed as ratios of non-cholesterol sterol to total cholesterol, were used as validated biomarkers of intestinal cholesterol absorption (β- sitosterol, cholestanol, campesterol), and endogenous cholesterol synthesis (lathosterol, desmosterol)(Reference Mashnafi, Plat and Mensink3). Non-cholesterol sterols were measured in plasma samples collected at the end of each diet, using GC-MS and epicoprostanol (5b-cholestan-3a-ol) as an internal standard. Data was analysed by a linear mixed model, with age, BMI, baseline visit, diets (high SFA, low SFA), and study centre included as fixed effects, and participants as a random effect (R version 4.1.2). Replacement of dietary SFA with UFA produced increases of between 15 to 35% in all three plasma biomarkers of intestinal cholesterol absorption (high vs lower SFA diet). Mean cholesterol-standardised ratios (95% CI) were as follows: β-sitosterol 0.95 (0.94 −1.01) vs 1.31 (1.27–1.34); cholestanol 1.25 (1.22–1.28) vs 1.44 (1.41–1.47); campesterol 0.91 (0.87–0.94) vs 1.14 (1.11–1.18) all p < 0.0001. Plasma desmosterol increased (0.61 (0.59–0.63) vs 0.66 (0.65–0.68)) p < 0.0001, while plasma lathosterol showed no significant change. In conclusion, the consistent increase in biomarkers of intestinal cholesterol absorption is in accord with the replacement of dietary SFA with UFA reducing the endogenous synthesis of cholesterol. However, since there was no evidence of a consistent dietary effect on the biomarkers of endogenous cholesterol synthesis, this fails to support a reciprocal relationship between cholesterol synthesis and absorption. Further insight into these effects will be gained through the stable isotope trace-labelling of dietary saturated fat in a follow-up study (RISSCI-2).

Tetracosahexaenoic acid (24:6ω-3) is an intermediate in the conversion of 18:3ω-3 to 22:6ω-3 in mammals. There is limited information about whether cells can assimilate and metabolize exogenous 24:6ω-3. This study compared the effect of incubation with 24:6ω-3 on the fatty acid composition of two related cell types, primary CD3+ T lymphocytes and Jurkat T cell leukemia, which differ in the integrity of the polyunsaturated fatty acid (PUFA) biosynthesis pathway. 24:6ω-3 was only detected in either cell type when cells were incubated with 24:6ω-3. Incubation with 24:6ω-3 induced similar increments in the amount of 22:6ω-3 in both cell types and modified the homeoviscous adaptations fatty acid composition induced by activation of T lymphocytes. The effect of incubation with 18:3ω-3 compared to 24:6ω-3 on the increment in 22:6ω-3 was tested in Jurkat cells because primary T cells cannot convert 18:3ω-3 to 22:6ω-3. The increment in the 22:6ω-3 content of Jurkat cells incubated with 24:6ω-3 was 19.5-fold greater than that of cells incubated with 18:3ω-3. Acyl-coA oxidase siRNA knockdown decreased the amount of 22:6ω-3 and increased the amount of 24:6ω-3 in Jurkat cells. These findings show exogenous 24:6ω-3 can be incorporated into primary human T lymphocytes and Jurkat cells and induces changes in fatty acid composition consistent with its conversion to 22:6ω-3 via a mechanism involving peroxisomal β-oxidation that is regulated independently from the integrity of the upstream PUFA synthesis pathway. One further implication is that consuming 24:6ω-3 may be an effective alternative means of achieving health benefits attributed to 20:5ω-3 and 22:6ω-3.

Dietary sugars are linked to the development of non-alcoholic fatty liver disease (NAFLD) and dyslipidaemia, but it is unknown if NAFLD itself influences the effects of sugars on plasma lipoproteins. To study this further, men with NAFLD (n=11) and low liver fat ‘controls’ (n= 14) were fed two iso-energetic diets, high or low in sugars (26% or 6% total energy) for 12 weeks, in a randomised, cross-over design. Fasting plasma lipid and lipoprotein kinetics were measured after each diet by stable isotope trace-labelling. There were significant differences in the production and catabolic rates of VLDL subclasses between men with NAFLD and controls, in response to the high and low sugar diets. Men with NAFLD had higher plasma concentrations of VLDL1-triacylglycerol (TAG) after the high (P

The reproducibility of clinical trial results is of major importance in nutrition research. Variability in the results of apparently similar dietary studies has been previously reported(Reference Sorkin, Kuszak and Williamson1), due to differences in study design, assessment methods, participants’ background diet and inter-individual variability. However, comparisons of identical dietary interventions performed in the same participants, which will control for some of the mentioned variability, are rare. This study aims to assess the reproducibility of an identical dietary intervention (Reading, Imperial, Surrey Saturated fat Cholesterol Intervention study 1 and 2, RISSCI-1 and RISSCI-2) performed on two occasions, on the outcomes of fasted lipids.

Context GLP-1 agonists control postprandial glucose and lipid excursion in type 2 diabetes; however the mechanism(s) are unclear. Objective To determine the mechanism(s) of postprandial lipid and glucose control with lixisenatide (GLP-1 analogue) in type 2 diabetes. Design Randomised, double-blind, cross-over study. Setting Centre for Diabetes, Endocrinology, and Research, Royal Surrey County Hospital, Guildford, UK Patients Eight obese men with type 2 diabetes (57.3±1.9yrs; BMI 30.3±1.0kg/m2, HbA1C 66.5±2.6mmol/mol, [8.2±0.3%]). Interventions Two metabolic studies, four-weeks after lixisenatide or placebo; with cross-over and repetition of studies. Main outcome measures Study one: very-low density lipoprotein (VLDL) and chylomicron (CM) triacylglycerol (TAG) kinetics were measured with iv bolus of [2H5]glycerol in a 12h study, with hourly feeding. Oral [13C]triolein, in a single meal, labelled enterally-derived TAG. Study two: glucose kinetics were measured with [U-13C]glucose in a mixed-meal (plus acetaminophen to measure gastric emptying) and variable iv [6,6-2H2]glucose infusion. Results Study one: CM-TAG (but not VLDL-TAG) pool-size, was lower with lixisenatide (P=0.046). Lixisenatide reduced CM [13C]oleate AUC60-480min concentration (P=0.048) and increased CM-TAG clearance; with no effect on CM-TAG production rate. Study two: postprandial glucose and insulin AUC0-240min were reduced with lixisenatide (P=0.0051, P˂0.05). Total glucose production rate (Ra) (P=0.015), Rameal (P=0.0098) and acetaminophen AUC0-360min (P=0.006) were lower with lixisenatide than placebo. Conclusions Lixisenatide reduced [13C]oleate concentration, derived from a single meal in CM-TAG, as well as glucose Rameal, through delayed gastric emptying. However day-long CM production, measured with repeated meal-feeding, was not reduced by lixisenatide and decreased CM-TAG concentration was due to increased CM-TAG clearance.

Resistant starch (RS) is classed as a dietary fiber and does not contribute to the postprandial glucose excursion. Two types may be formed during food processing: RS3 when a starchy carbohydrate cools; RS5 when starch is heated in the presence of lipid. It is unknown whether RS5 can be produced under domestic cooking conditions or whether different fat types would affect the amount formed. Nor is it known whether sufficient RS3 remains after a chilled meal is reheated to have a significant impact on the postprandial glucose response. The aim of this study was to determine if different solid fat spreads (of varying fatty acid composition) incorporated into a mashed potato meal, served either freshly cooked or chilled and reheated, would produce different effects on the postprandial glucose and insulin responses. Ten healthy adults (age: 29 ± 5 y; BMI: 21.7 ± 2.6 kg/m2) attended six study days in a randomized crossover design. On each day, they consumed mashed potatoes (203 g) prepared with one of three solid fat spreads (butter, sunflower oil, olive oil, each providing 20 g fat). Each meal was consumed freshly cooked or microwave-reheated (after two days refrigeration at 4°C). Capillary blood samples were taken for 180 min postprandially. Plasma glucose and insulin were measured by glucose-oxidase method and ELISA respectively. Glucose incremental area under the curve (IAUC) was significantly lower for the reheated meal with butter, compared to the equivalent freshly cooked meal (P = 0.030). Insulinogenic Index (IGI), a surrogate measure of first phase insulin response, was significantly lower for the freshly cooked butter meal in comparison with the reheated equivalent (P = 0.031). There were no other differences between meals, either for fat type or preparation method. Differences in RS formation may explain these results; work is underway to measure both total RS and RS5. Other possible explanations are differences in effects on glucose absorption, such as via delayed gastric emptying. Whilst a beneficial effect on postprandial glucose and IGI was observed for the reheated butter meal, in comparison to the freshly cooked, it should be remembered that butter contains saturated fat, which has detrimental effects on blood lipids and should only be consumed in moderation. Biotechnology and Biological Sciences Research Council, UK.

Purpose: UK guidelines recommend dietary saturated fatty acids (SFAs) should not exceed 10% total energy (%TE) for cardiovascular disease prevention, with benefits observed when SFAs are replaced with unsaturated fatty acids (UFAs). This study aimed to assess the efficacy of a dietary exchange model using commercially available foods to replace SFAs with UFAs. Methods: Healthy men (n=109, age 48, SD 11y) recruited to the Reading, Imperial, Surrey, Saturated fat Cholesterol Intervention-1 (RISSCI-1) study (ClinicalTrials.Gov n°NCT03270527) followed two sequential 4-week isoenergetic moderate-fat (34%TE) diets: high-SFA (18%TE SFAs, 16%TE UFAs) and low-SFA (10%TE SFAs, 24%TE UFAs). Dietary intakes were assessed using 4-day weighed diet diaries. Nutrient intakes were analysed using paired t-tests, fasting plasma phospholipid fatty acid (PL-FA) profiles and dietary patterns were analysed using orthogonal partial least square discriminant analyses. Results: Participants exchanged 10.2%TE (SD 4.1) SFAs for 9.7%TE (SD 3.9) UFAs between the high and low-SFA diets, reaching target intakes with minimal effect on other nutrients or energy intakes. Analyses of dietary patterns confirmed successful incorporation of recommended foods from commercially available sources (e.g. dairy products, snacks, oils, and fats), without affecting participants’ overall dietary intakes. Analyses of plasma PL-FAs indicated good compliance to the dietary intervention and foods of varying SFA content. Conclusions: RISSCI-1 dietary exchange model successfully replaced dietary SFAs with UFAs in free-living healthy men using commercially available foods, and without altering their dietary patterns. Further intervention studies are required to confirm utility and feasibility of such food-based dietary fat replacement models at a population level.

Objective. To investigate the mechanism for increased ketogenesis following treatment with SGLT2 inhibitor, dapagliflozin in people with type 2 diabetes. Research, Design & Methods. This was a double-blind placebo-controlled crossover study with a 4-week washout period. Participants received dapagliflozin or placebo in random order for 4 weeks. After each treatment, they ingested 30ml of olive oil containing [U-13C] palmitate to measure ketogenesis with blood sampling for 480 min. Stable isotopes of glucose and glycerol were infused to measure glucose flux and lipolysis respectively at 450-480 min. Results. Glucose excretion rate was higher and peripheral glucose uptake lower with dapagliflozin than placebo. Plasma beta-hydroxybutyrate (BOHB) concentrations and [13C2] BOHB concentrations were higher and glucose concentrations lower with dapagliflozin than placebo. Non-esterified fatty acids (NEFA) were higher with dapagliflozin at 300 and 420 min but lipolysis at 450-480 min was not different. Triacylglycerol (TAG) at all time points and endogenous glucose production rate at 450-480 min were not different between treatments. Conclusions. The increase in ketone enrichment from the ingested palmitic acid tracer suggests meal derived fatty acids contribute to the increase in ketones during treatment with dapagliflozin. The increase in BOHB concentration with dapagliflozin, occurred with only minimal changes in plasma NEFA concentration and no change in lipolysis. This suggests a metabolic switch to increase ketogenesis within the liver.

Potatoes have been an affordable, staple part of the diet for many hundreds of years. Recently however, there has been a decline in consumption, perhaps influenced by erroneous reports of being an unhealthy food. This review provides an overview of the nutritional value of potatoes and examines the evidence for associations between potato consumption and non-communicable diseases. Potatoes are an important source of micronutrients, such as vitamin C, vitamin B6, potassium, folate, and iron and contribute a significant amount of fibre to the diet. However, nutrient content is affected by cooking method; boiling causes leaching of water-soluble nutrients, whereas frying can increase the resistant starch content of the cooked potato. Epidemiological studies have reported associations between potato intake and obesity, type 2 diabetes and cardiovascular disease. However, results are contradictory and confounded by lack of detail on cooking methods. Indeed, potatoes have been reported to be more satiating than other starchy carbohydrates, such as pasta and rice, which may aid weight maintenance. Future research should consider cooking methods in the study design in order to reduce confounding factors and further explore the health impact of this food.

Gastric emptying (GE) is the process of food being processed by the stomach and delivered to the small intestine where nutrients such as lipids are absorbed into the blood circulation. The combination of an easy and inexpensive method to measure GE such as the CO2 breath test using the stable isotope [ 13 C]octanoic acid with semi-mechanistic modelling could foster a wider application in nutritional studies to further understand the metabolic response to food. Here, we discuss the use of the [ 13 C]octanoic acid breath test to label the solid phase of a meal, and the factors that influence GE to support mechanistic studies. Furthermore, we give an overview of existing mathematical models for the interpretation of the breath test data and how much nutritional studies could benefit from a physiological based pharmacokinetic model approach.

Mycobacterium tuberculosis (Mtb) infects macrophages and macrophage-derived foam cells, a hallmark of granulomata in tuberculous lesions. We analysed the effects of lipid accumulation in human primary macrophages and quantified strong triglyceride and phospholipid remodelling which depended on the dietary fatty acid used for the assay. The enrichment of >70% in triglyceride and phospholipids can alter cell membrane properties, signalling and phagocytosis in macrophages. In conventional macrophage cultures, cells are heterogeneous, small or large macrophages. In foam cells, a third population of 30% of cells with increased granularity can be detected. We found that foam cell formation is heterogenous and that lipid accumulation and foam cell formation reduces the phagocytosis of Mtb. Under the conditions tested, cell death was highly prevalent in macrophages, whereas foam cells were largely protected from this effect. Foam cells also supported slower Mtb replication, yet this had no discernible impact on the intracellular efficacy of four different antitubercular drugs. Foam cell formation had a significant impact in the inflammatory potential of the cells. TNF-α, IL-1β and prototypical chemokines were increased. The ratio of inflammatory IL-1β, TNF-α and IL-6 versus anti-inflammatory IL-10 was significantly higher in response to Mtb versus LPS, and was increased in foam cells compared to macrophages, suggestive of increased pro-inflammatory properties. Cytokine production correlated with NF-κB activation in our models. We conclude that foam cell formation reduces the host cell avidity for, and phagocytosis of, Mtb while protecting the cells from death. This protective effect is associated with enhanced inflammatory potential of foam cells and restricted intracellular growth of Mtb.

Background Epidemiological studies suggest that metformin may reduce the incidence of cancer in patients with diabetes and multiple late phase clinical trials assessing the potential of repurposing this drug are underway. Transcriptomic profiling of tumour samples is an excellent tool to understand drug bioactivity, identify candidate biomarkers and assess for mechanisms of resistance to therapy. Methods Thirty-six patients with untreated primary breast cancer were recruited to a window study and transcriptomic profiling of tumour samples carried out before and after metformin treatment. Results Multiple genes that regulate fatty acid oxidation were upregulated at the transcriptomic level and there was a differential change in expression between two previously identified cohorts of patients with distinct metabolic responses. Increase in expression of a mitochondrial fatty oxidation gene composite signature correlated with change in a proliferation gene signature. In vitro assays showed that, in contrast to previous studies in models of normal cells, metformin reduces fatty acid oxidation with a subsequent accumulation of intracellular triglyceride, independent of AMPK activation. Conclusions We propose that metformin at clinical doses targets fatty acid oxidation in cancer cells with implications for patient selection and drug combinations. Clinical Trial Registration NCT01266486.

BACKGROUND Duodenal mucosal resurfacing (DMR) is a novel day-case endoscopic intervention which results in weight loss-independent reductions in HbA1c in patient with type 2 diabetes mellitus (T2DM). We hypothesized that DMR works by increasing insulin sensitivity and we aimed to investigate the mechanism of action of DMR through longitudinal metabolic phenotyping in humans. METHODSThirty-two insulin-resistant women with polycystic ovary syndrome (PCOS) and obesity were randomised in a double-blinded manner to DMR or sham endoscopy. They underwent measurements of insulin sensitivity using euglycaemic hyperinsulinaemic clamps, insulin secretion using oral glucose tolerance tests and reproductive function using weekly reproductive hormone profiles and ovarian ultrasonography for 6 months post-intervention. RESULTSA small increase in total body insulin sensitivity measured by the clamp was observed in both groups at week 12. An increase in insulin sensitivity, as measured by HOMA-IR, was observed in both groups at week 24. There was an increase in the number of menses (median 2 DMR, 0.5 sham). There were no significant differences between the two groups in these outcomes or insulin secretion. CONCLUSIONSThese findings suggest that DMR does not work by increasing insulin sensitivity in euglycaemic, insulin resistant women with PCOS. The procedure may exert its effects only in the context of hyperglycaemia or pathologically hyperplastic, insulin-desensitised duodenal mucosa.

Background: Epidemiological studies suggest that metformin may reduce the incidence of cancer in patients with diabetes and multiple late phase clinical trials assessing the potential of repurposing this drug are underway. Transcriptomic profiling of tumour samples is an excellent tool to understand drug bioactivity, identify candidate biomarkers and assess for mechanisms of resistance to therapy. Methods: 36 patients with untreated primary breast cancer were recruited to a window study and transcriptomic profiling of tumour samples carried out before and after metformin treatment. Results: Multiple genes that regulate fatty acid oxidation were upregulated at the transcriptomic level and there was a differential change in expression between 2 previously identified cohorts of patients with distinct metabolic responses. Increase in expression of a mitochondrial fatty oxidation gene composite signature correlated with change in a proliferation gene signature. In vitro assays showed that, in contrast to previous studies in models of normal cells, metformin reduces fatty acid oxidation with a subsequent accumulation of intracellular triglyceride, independent of AMPK activation. Conclusions: We propose that metformin at clinical doses targets fatty acid oxidation in cancer cells with implications for patient selection and drug combinations.

Menopause increases the risk of cardiovascular disease (CVD) which in part has been attributed to the rise in cholesterol and blood pressure (BP). This study examined the hypothesis that menopausal changes in body composition and regional fat depots relate to the change in CVD risk factors. A prospective recall study was designed to capture premenopausal women to be re-examined soon after menopause. A total of 97 women from the Oxford Biobank underwent dual x-ray absorptiometry, blood biochemistry, and BP readings pre- and postmenopause. Despite minimal changes in body weight over the 5.1 ± 0.9 year follow-up period, there was an increase in total fat mass and a decline in lean mass, where the proportional change of regional fat mass was the greatest for the visceral fat depot (+22%, P 

Despite opposing insulin sensitivity and cardiometabolic risk, both athletes and patients with type 2 diabetes have increased skeletal myocyte fat storage: the so-called "athlete's paradox". In a parallel non-randomised, non-blinded trial (NCT03065140), we characterised and compared the skeletal myocyte lipid signature of 29 male endurance athletes and 30 patients with diabetes after undergoing deconditioning or endurance training respectively. The primary outcomes were to assess intramyocellular lipid storage of the vastus lateralis in both cohorts and the secondary outcomes were to examine saturated and unsaturated intramyocellular lipid pool turnover. We show that athletes have higher intramyocellular fat saturation with very high palmitate kinetics, which is attenuated by deconditioning. In contrast, type 2 diabetes patients have higher unsaturated intramyocellular fat and blunted palmitate and linoleate kinetics but after endurance training, all were realigned with those of deconditioned athletes. Improved basal insulin sensitivity was further associated with better serum cholesterol/triglycerides, glycaemic control, physical performance, enhanced post insulin receptor pathway signalling and metabolic sensing. We conclude that insulin-resistant, maladapted intramyocellular lipid storage and turnover in patients with type 2 diabetes show reversibility after endurance training through increased contributions of the saturated intramyocellular fatty acid pools. Clinical Trial Registration: NCT03065140: Muscle Fat Compartments and Turnover as Determinant of Insulin Sensitivity (MISTY).

Impaired adipose tissue insulin signalling is a critical feature of insulin resistance. Here we identify a pathway linking the lipolytic enzyme hormone-sensitive lipase (HSL) to insulin action via the glucose-responsive transcription factor ChREBP and its target, the fatty acid elongase ELOVL6. Genetic inhibition of HSL in human adipocytes and mouse adipose tissue results in enhanced insulin sensitivity and induction of ELOVL6. ELOVL6 promotes an increase in phospholipid oleic acid, which modifies plasma membrane fluidity and enhances insulin signalling. HSL deficiency–mediated effects are suppressed by gene silencing of ChREBP and ELOVL6. Mechanistically, physical interaction between HSL, independent of lipase activity, and the isoform activated by glucose metabolism ChREBPα impairs ChREBPα translocation into the nucleus and induction of ChREBPβ, the isoform with high transcriptional activity that is strongly associated with whole-body insulin sensitivity. Targeting the HSL–ChREBP interaction may allow therapeutic strategies for the restoration of insulin sensitivity.

Abstract only Background: Increased intramyocellular lipid storage occurs in skeletal muscle of both athletes and type 2 diabetes patients (T2D) but their metabolic phenotypes are at opposite ends of insulin sensitivity and cardiometabolic risk. Hypothesis: Intramyocellular lipid saturation and flux are determinants of insulin resistance. Methods: Age-matched athletes (n=28) and T2D (n=27) underwent 1 H-magnetic resonance spectroscopy for analysis of saturated (fSL) and unsaturated lipid (fUL) fractions of vastus lateralis, blood lipids, glucose and insulin sensitivity, skeletal muscle biopsy for lipidomics/insulin signalling, cardiopulmonary exercise peak VO 2 and intravenous administration of stable isotopes of [U- 13 C]-palmitate and -linoleate for skeletal muscle saturated/unsaturated fatty acid fluxes (fractional synthesis rate, FSR). Tests were performed at baseline and after controlled insulin sensitivity changes (4-week athletes deconditioning and endurance exercise training at ≥65% of baseline peakVO 2, 5 hours/week x 8 weeks in T2D). Results: We demonstrate higher intramyocellular fSL and lower fUL in athletes compared to T2D (p=0.0004). Trained athletes have a 4-fold (palmitate) and 2.2-fold (linoleate) higher FSR compared to T2D (p=0.005 and 0.006, respectively). Athletes’ deconditioning results in reductions in skeletal muscle lipid saturation, palmitate and linoleate FSR. Conversely, 8-week endurance training of T2D results in intramyocellular lipid saturation increase (p

Purpose of review: There is considerable political and public awareness of new recommendations to reduce sugars and sugar-sweetened beverages in our diets. It is therefore timely to review the most recent changes in guidelines, with a focus on evidence for metabolic health, recent research in the area and gaps in our knowledge. Recent findings: Sufficient evidence links a high intake of sugar to dental caries and obesity, and high intakes of sugar-sweetened beverages in particular to increased risk of type 2 diabetes. This has led to the updating of dietary recommendations related to added sugars in the diet. The effects of specific sugars at usual intakes as part of an isoenergetic diet are less clear. The glycaemic response to food is complex and mediated by many factors, but sugar intake is not necessarily the major component. Summary: There are many challenges faced by healthcare professionals and government bodies in order to improve the health of individuals and nations through evidence-based diets. Sufficiently powered long-term mechanistic studies are still required to provide evidence for the effects of reducing dietary sugars on metabolic health. However, there are many challenges for research scientists in the implementation of these studies.

Background: Observational studies often infer hepatic de novo lipogenesis (DNL) by measuring circulating fatty acid (FA) markers; however it remains to be elucidated whether these markers accurately reflect hepatic DNL. Objective: We investigated associations between fasting hepatic DNL and proposed FA markers of DNL in subjects consuming their habitual diet. Design: Fasting hepatic DNL was assessed using ²H2O (heavy water) in 149 non-diabetic men and women and measuring the synthesis of very low-density lipoprotein triglyceride (VLDL-TG) palmitate. FA markers of blood lipid fractions was determined by gas chromatography (GC). Results: Neither the lipogenic index (16:0/18:2n-6) nor the SCD index (16:1n-7/16:0) in VLDL-TG were associated with isotopically assessed DNL (r=0.13, P=0.1 and r=-0.08, P=0.35, respectively). The relative abundance (mol%) of 14:0, 16:0 and 18:0 in VLDL-TG were weakly (r≤0.35) associated with DNL whereas abundance of 16:1n-7, 18:1n-7 and 18:1n-9 were not associated. When the cohort was split by median DNL, only abundance of 14:0 and 18:0 in VLDL-TG could discriminate between subjects having high (11.5%) and low (3.8%) fasting hepatic DNL. Based on a subgroup, FA markers in total plasma TG, plasma cholesteryl esters, plasma phospholipids and red blood cell phospholipids were generally not associated with DNL. Conclusions: The usefulness of circulating FAs as markers of hepatic DNL in healthy individuals consuming their habitual diet is limited due to their inability to clearly discriminate between individuals with low and high fasting hepatic DNL.

Polyunsaturated fatty acids (PUFAs) are important for immune function. Limited evidence indicates that immune cell activation involves endogenous PUFA synthesis, but this has not been characterised. To address this, we measured metabolism of 18:3n-3 in quiescent and activated peripheral blood mononuclear cells (PBMCs), and in Jurkat T cell leukaemia. PBMCs from men and women (n = 34) were incubated with [1-13C]18:3n-3 with or without Concanavalin A (Con. A). 18:3n-3 conversion was undetectable in unstimulated PBMCs, but up-regulated when stimulated. The main products were 20:3n-3 and 20:4n-3, while 18:4n-3 was undetectable, suggesting initial elongation and 8 desaturation. PUFA synthesis was 17.4-fold greater in Jurkat cells than PBMCs. The major products of 18:3n-3 conversion in Jurkat cells were 20:4n-3, 20:5n-3 and 22:5n-3. 13C Enrichment of 18:4n-3 and 20:3n-3 suggests parallel initial elongation and Δ6 desaturation. The FADS2 inhibitor SC26196 reduced PBMC, but not Jurkat cell, proliferation suggesting PUFA synthesis is involved in regulating mitosis in PBMCs. Con. A stimulation increased FADS2, FADS1, ELOVL5 and ELOVL4 mRNA expression in PBMCs. A single transcript corresponding to the major isoform of FADS2, FADS20001, was detected in PBMCs and Jurkat cells. PBMC activation induced hypermethylation of a 470bp region in the FADS2 5’-regulatory sequence. This region was hypomethylated in Jurkat cells compared to quiescent PBMCs. These findings show that PUFA synthesis involving initial elongation and 8 desaturation is involved in regulating PBMC proliferation and is regulated via transcription possibly by altered DNA methylation. These processes were dysregulated in Jurkat cells. This has implications for understanding the regulation of mitosis in normal and transformed lymphocytes.

The menopause is accompanied by increased risk of obesity, altered body fat distribution and decreased skeletal muscle mass. The resulting decrease in RMR should be accompanied by a compensatory change in energy balance to avoid weight gain. We aimed to investigate habitual energy intake and expenditure in pre- and postmenopausal women matched for abdominal obesity. We recruited fifty-one healthy Caucasian women, BMI > 18·5 and 

Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that endothelial FABP4 is induced by the NOTCH1 signalling pathway, which is involved in mechanisms of resistance to anti-angiogenic tumour therapy1. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability, and angiogenesis. Through therapeutic delivery of siRNA targeting murine FABP4, we investigated the effect of stromal FABP4 knockdown on tumour growth and blood vessel formation. In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species (ROS), and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to increased expression of endothelial FABP4 that decreased when NOTCH1 and VEGFA inhibitors were used in combination. Angiogenesis, growth, and metastasis in ovarian tumour xenografts were markedly inhibited by therapeutic siRNA delivery targeting mouse FABP4. Therapeutic targeting of endothelial FABP4 by siRNA in vivo has anti-angiogenic and anti-tumour effects with minimal toxicity and should be investigated further.

Objectives: To comprehensively survey the sugar and nutrient contents of yogurt products available in UK supermarkets, in particular those marketed to children. Design: A cross-sectional survey of yogurt products available in the UK’s supermarkets in November 2016. Methods: Data were collected from five major online UK supermarkets and a process flow strategy was used to place yogurts into eight categories: children’s, dairy alternatives, dessert, drinks, fruit, flavoured, natural/Greek style and organic. A comprehensive database of product information for 921 unique products was created and analysed. Results: The total sugar, fat, protein, calcium and energy contents were highly variable across categories, and the ranges were extremely broad. Although lower than the dessert category, the medians (range) of the total sugar content of children’s (10.8 g/100 g (4.8–14.5)), fruit (11.9 g/100 g (4.6–21.3)), flavoured (12.0 g/100 g (0.1–18.8)) and organic (13.1 g/100 g (3.8–16.9)) yogurt products were all well above 10 g/100 g, and represented >45% of total energy. Only two out of 101 children’s yogurt and fromage frais products surveyed qualified as low sugar (≤5 g/100 g). Natural/Greek yogurts had dramatically lower sugar contents (5.0 g/100 g (1.6, 9.5), largely lactose) than all other categories. While low-fat (

The menopause is accompanied by increased risk of obesity, altered body fat distribution, and decreased skeletal muscle mass. The resulting decrease in RMR should be accompanied by a compensatory change in energy balance to avoid weight gain. We aimed to investigate habitual energy intake and expenditure in pre- and post-menopausal women matched for abdominal obesity. We recruited fifty-one healthy Caucasian women, BMI >18.5 and < 35, aged 35 - 45 years (pre-menopausal, n=26) and 55-65 years (post-menopausal, n=25). Energy intake was measured using 3-day diet diaries and dietary fat quality assessed using adipose tissue fatty acid biomarkers. RMR was measured using indirect calorimetry, and total (TEE) and activity energy expenditure using a combined accelerometer and heart rate monitor. Post-menopausal women had lower RMR and TEE and spent significantly less time undertaking moderate exercise than pre-menopausal women. Post-menopausal women had a tendency for a lower calorie intake, and a similar macronutrient intake but a significantly lower adipose tissue n-6/n-3 index (24.6 (1.6) v 37.7 (3.1), P

Background Experimental data suggest that apolipoprotein (apo) C-II and C-III regulate triglyceride-rich lipoprotein (TRL) metabolism, but there are limited studies in humans. We investigated the metabolic associations of TRLs with apoC-II and apoC-III concentrations and kinetics in women. Material and methods The kinetics of plasma apoC-II, apoC-III and very low-density lipoprotein (VLDL) apoB-100 and triglycerides were measured in the postabsorptive state using stable isotopic techniques and compartmental modeling in 60 women with wide-ranging body mass index (19.5-32.9kg/m2). Results Plasma apoC-II and apoC-III concentrations were positively associated with the concentration of plasma triglycerides, VLDL1- and VLDL2- apoB-100 and triglyceride (all P

Background Android fat distribution (abdominal obesity) is associated with insulin resistance, hepatic steatosis and greater secretion of large very low density lipoprotein (VLDL) particles in men. Since abdominal obesity is becoming increasingly prevalent in women we aimed to investigate the relationship between android fat and hepatic lipid metabolism in pre- and post-menopausal women. Methods and Results We used a combination of stable isotope tracer techniques to investigate intrahepatic fatty acid synthesis and partitioning in 29 lean and 29 abdominally obese women (android fat/total fat 0.065 (0.02-0.08) and 0.095 (0.08-0.11) respectively). Thirty women were pre-menopausal aged 35-45 and they were matched for abdominal obesity with 28 post-menopausal women aged 55-65. As anticipated, abdominal obese women were more insulin resistant with enhanced hepatic secretion of large (404±30 v 268±26 mg/kg lean mass, P

We hypothesised that probiotic supplementation (PRO) increases the absorption and oxidation of orally ingested maltodextrin during 2h endurance cycling, thereby sparing muscle glycogen for a subsequent time trial (simulating a road race). Measurements were made of lipid and carbohydrate oxidation, plasma metabolites and insulin, gastrointestinal permeability, and subjective symptoms of discomfort. Seven male cyclists were randomized to PRO (bacterial composition given in methods) or placebo (PLC) for four weeks, separated by a 14-day washout period. After each period, cyclists consumed a 10% maltodextrin solution (initial 8 mL·kg-1 bolus and 2 mL·kg-1 every 15 min) while exercising for 2h at 55% Wmax followed by a 100 kJ time trial. PRO resulted in small increases in peak oxidation rates of the ingested maltodextrin (0.84 ± 0.10 vs 0.77 ± 0.09 g·min-1, P = 0.016), and mean total carbohydrate oxidation (2.20 ± 0.25 vs 1.87 ± 0.39 g·min-1, P = 0.038), while fat oxidation was reduced (0.40 ± 0.11 vs 0.55 ± 0.10 g·min-1, P = 0.021) . During PRO small but significant increases were seen in glucose absorption, plasma glucose and insulin concentration and decreases in NEFA and glycerol. Differences between markers of GI damage and permeability and time trial performance were not significant (P > 0.05). In contrast to the hypothesis, PRO led to minimal increases in absorption and oxidation of the ingested maltodextrin and small reductions in fat oxidation, while having no effect on subsequent time trial performance.

Most postprandial studies have investigated the response of a single meal, yet the ingestion of sequential meals is more typical in a Western society. The aim of this review is to explain how natural and stable isotope tracers of fatty acids have been used to investigate the metabolism of dietary fat after single and multiple meals, with a focus on in vivo measurements of adipose tissue metabolism. When stable isotope tracers are combined with arteriovenous difference measurements, very specific measurements of metabolic flux across tissues can be made. We have found that adipose tissue is a net importer of dietary fat for 5 h following a single test meal and for most of the day during a typical three-meal eating pattern. When dietary fat is cleared from plasma, some fatty acids ‘spillover’ into the plasma and contribute up to 50 % of postprandial plasma non-esterified fatty acid concentrations. Therefore, plasma non-esterified fatty acid concentrations after a meal reflect the balance between intracellular and extracellular lipolysis in adipose tissue. This balance is altered after the acute ingestion of fructose. The enzyme lipoprotein lipase is a key modulator of fatty acid flux in adipose tissue and its rate of action is severely diminished in obese men. In conclusion, in vivo studies of human metabolism can quantify the way that fatty acid trafficking modulates plasma lipid concentrations. The magnitude of fatty acid flux from adipose tissue has implications for ectopic fat deposition in tissues such as the liver and muscle.

Specialized metabolic-sensors in the hypothalamus regulate blood glucose levels by influencing hepatic glucose output and hypoglycemic counter regulatory responses. Hypothalamic reactive oxygen species (ROS) may act as a metabolic signal mediating responses to changes in glucose, other substrates and hormones. The role of ROS in the brain’s control of glucose homeostasis remains unclear. We hypothesized that hydrogen peroxide (H2O2), a relatively stable form of ROS, acts as a sensor of neuronal glucose consumption and availability and that lowering brain H2O2 with the enzyme catalase would lead to systemic responses increasing blood glucose. During hyperinsulinemic euglycemic clamps in rats, ICV catalase infusion resulted in increased hepatic glucose output, which was associated with reduced neuronal activity in the arcuate nucleus of the hypothalamus (ARC). Electrophysiological recordings revealed a subset of ARC neurons expressing pro-opiomelanocortin (POMC) that were inhibited by catalase and excited by H2O2. During hypoglycemic clamps, ICV catalase increased glucagon and epinephrine responses to hypoglycemia, consistent with perceived lower glucose levels. Our data suggest that H2O2 represents an important metabolic cue which, through tuning the electrical activity of key neuronal populations such as POMC neurons, may have a role in the brain’s influence of glucose homeostasis and energy balance.

Different lipid fractions in humans have characteristic fatty acid profiles and these are maintained partly through diet and to a lesser extent through endogenous synthesis. The enzyme stearoyl-CoA desaturase (SCD; EC 1.14.99.5) is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids such as palmitoleic acid (16:1 n-7) and oleic acid (18:1 n-9). These are the two most abundant monounsaturated fatty acids in human plasma lipids, membranes and adipose tissue. Although in quantitative terms, the endogenous synthesis of fatty acids in humans is not great in most circumstances, it is becoming increasingly evident that SCD plays important structural and metabolic roles. In addition, 16:1 n-7 has been purported to act as a beneficial 'lipokine' in an animal model. Research in humans has relied on indirect measurements of SCD1 activity and therefore, much of our understanding has come from work on animal models. However, results have been somewhat counterintuitive and confusing, so the purpose of this review is to try to summarise our current understanding of this fascinating enzyme.

Purpose of review In 2004, the ‘omega-3 index’ was described as the sum of eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) in red blood cells (RBCs) as an index of coronary heart disease mortality. This review outlines new evidence to support the omega-3 index as a tool to inform disease prognosis. Recent findings Recent studies have reported differential metabolism of EPA and DHA. High dose supplementation with EPA and DHA led to increased levels of RBC DHA that were associated with decreased liver fat. EPA and DHA in RBCs were associated with reduced mortality in a prospective study of patients with cardiac disease; the strongest association was with EPA. A diet containing 9.5 g alpha linolenic acid lead to an increase in EPA but not DHA status in middle aged women. Summary: Dietary intake or supplementation studies with n-3 fatty acids should include measurement of n-3 status in a standardised way. The omega-3 index, reflecting EPA and DHA status throughout the body, is convenient and may be appropriate in some cases, but since EPA and DHA assimilate differently in membranes, and have different potency, measurement of individual fatty acid composition in RBCs may be more informative

The expansion of lower-body adipose tissue (AT) is paradoxically associated with reduced cardiovascular disease and diabetes risk. We examined whether the beneficial metabolic properties of lower-body AT are related to the production and release of the insulin-sensitizing lipokine palmitoleate (16:1n-7). Using venoarterial difference sampling, we investigated the relative release of 16:1n-7 from lower-body (gluteofemoral) and upper-body (abdominal subcutaneous) AT depots. Paired gluteofemoral and abdominal subcutaneous AT samples were analyzed for triglyceride fatty acid composition and mRNA expression. Finally, the triglyceride fatty acid composition of isolated human preadipocytes was determined. Relative release of 16:1n-7 was markedly higher from gluteofemoral AT compared with abdominal subcutaneous AT. Stearoyl-CoA desaturase 1 (SCD1), the key enzyme involved in endogenous 16:1n-7 production, was more highly expressed in gluteofemoral AT and was associated with greater enrichment of 16:1n-7. Furthermore, isolated human preadipocytes from gluteofemoral AT displayed a higher content of SCD1-derived fatty acids. We demonstrate that human gluteofemoral AT plays a major role in determining systemic concentrations of the lipokine palmitoleate. Moreover, this appears to be an inherent feature of gluteofemoral AT. We propose that the beneficial metabolic properties of lower-body AT may be partly explained by the intrinsically greater production and release of palmitoleate.

Scope: Fructose exacerbates post-prandial hypertriacylglycerolaemia. This may be partly due to increased enterocyte de novo lipogenesis (DNL). It is unknown whether this is concentration-dependent or whether fructose has a greater effect on lipid synthesis than glucose. The dose-dependent effects of fructose and glucose on DNL and de novo triacylglycerol (TAG)-glycerol synthesis were investigated in an enterocyte model; Caco-2 cells. Methods and results: Caco-2 cells were treated for 96h with 5mM, 25mM or 50mM fructose or glucose, or 12.5mM fructose/12.5mM glucose mix. DNL was measured following addition of [13C2]-acetate to apical media. In separate experiments, [13C6]-fructose and [13C6]-glucose were used to measure DNL and de novo TAG-glycerol synthesis. DNL from [13C2]-acetate was detected following all treatments, with greater amounts in intracellular than secreted (media) samples (all P

The purpose of this study was to determine whether or not partial acylglycerols, resulting from triacylglycerol hydrolysis, accumulate during the process of fat deposition in vivo. We measured mono-, di- and triacylglycerol levels in arterialized and adipose tissue venous plasma in normal subjects before and after a test meal. The mean concentrations of partial acylglycerols were consistently low in all plasma samples, accounting for less than 2% of the total acylglycerol measurement. The absence of monoacylglycerol accumulation in the adipose tissue venous plasma samples indicates that the disposal of monoacylglycerol by the adipose tissue was not rate limiting for complete hydrolysis of triacylglycerol in vivo. After heparin was injected intravenously 300 min after the high-fat meal, the mean concentration of monoacylglycerol in arterialized plasma increased from 1.6 ± 0.6 μmol/l to 106 ± 19 μmol/l (P = 0.01), whereas the mean concentration of diacylglycerol did not change (13.0 ± 2.3 μmol/l before, 17.4 ± 4.2 μmol/l after).

A sensitive method has been developed to measure specific mono-, di- and triacylglycerol concentrations in human plasma, using thin-layer chromatography and enzymatic assay. The levels of partial acylglycerols in human plasma from fasting subjects were lower than previous reports had suggested and amounted to less than 3% of the total acylglycerols. After heparin injection the plasma monoacylglycerol concentration increased markedly (P < 0.01) while the triacylglycerol concentration decreased significantly (P < 0.001). The plasma diacylglycerol concentration did not change significantly although it increased as a percentage of the total (P < 0.05); after heparin partial acylglycerols accounted for more than 10% of the total. After a high-fat meal the plasma concentrations of di- and triacylglycerol increased approximately two-fold (P < 0.005) but no significant change was observed for mono-acylglycerol. The percentage contribution of partial acylglycerols was unchanged (2.6% fasting, 2.4% postprandially).

There is net outward flow of fatty acids from adipose tissue in the fasted state but net inward flow and storage in the postprandial state. We investigated how this is regulated. Arteriovenous differences were measured across a subcutaneous adipose depot in six normal subjects before and for 5 h after a meal containing 80 g fat and 80 g carbohydrate. In five further experiments, insulin was infused at 40 mU · m -2 · min -1 from 30 min after the meal, clamping the plasma glucose. Net transcapillary fatty acid flow changed from negative (outward flow from tissue to capillaries) in the postabsorptive state to consistently positive (net inward flow, implying fat storage) after the meal despite continued net efflux of fatty acids into venous blood. In the 'clamped' experiments (with additional insulin), net fatty acid efflux in the venous blood was suppressed and positive transcapillary flux (storage) was more marked. Regulation of fatty acid flow appeared to depend on coordinated changes in hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL) action and fatty acid esterification. Additional insulin caused no further suppression of HSL or activation of LPL but markedly stimulated fatty acid retention (presumed to represent esterification). In the absence of additional insulin, a high proportion of the fatty acids liberated by LPL are released into the venous plasma in both postabsorptive and postprandial states. We hypothesize that this 'loss' of fatty acids is necessary to give precise control to the pathway of fat storage.