Dr Barbara Fielding

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.

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.

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 rate after the high sugar diet (P low sugar diet (P hepatic TAG into a higher production of VLDL1-TAG (P contrast, a higher production of VLDL2-TAG (P VLDL subclass kinetics could be explained, in part, by differences in the contribution of
fatty acids from intra-hepatic stores, and de novo lipogenesis. This study provides new
evidence that liver fat accumulation leads to a differential partitioning of hepatic TAG into
large and small VLDL subclasses, in response to high and low intakes of sugars.

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 D8 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 ”6 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 D8 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.

Conversion of the essential n-3 (18:3n-3) and n-6 (18:2n-6) fatty acids to longer chain polyunsaturated fatty acids (PUFA) involves sequential desaturation and elongation reactions. Previous studies have reported gender differences in n-3 PUFA synthesis, whereas the effect of age is less clear. n-3 PUFAs are reported to have important effects on immune cell function. A previous study reported long chain PUFA synthesis in mitogen stimulated but not quiescent peripheral blood mononuclear cells (PBMCs). However, the underlying mechanism is not known.

PUFA synthesis was investigated in PBMCs incubated with [1-13C]18:3n-3 for 48 h. Activation with the T-lymphocyte mitogen concanavalin A (Con A) increased PUFA synthesis. 22:6n-3 synthesis was not detected. [1-13C] incorporation was greatest for 20:3n-3 suggesting initial chain elongation is an important fate for 18:3n-3. Con A increased expression of three key genes (FADS2, FADS1 and ELOVL5) involved in PUFA synthesis, suggesting upregulation of the pathway is controlled at the transcriptional level. ELOVL2 expression was negligible, possibly explaining the lack of 22:6n-3 synthesis. Con A increased methylation of 12 CpGs in the FADS2 promoter contradicting the general view that DNA methylation represses transcription. Subsequent 5?RACE analysis verified that activated PBMCs were not using an alternative promoter for FADS2 transcription. Contrary to expectation, 18:3n-3 conversion in activated PBMCs was not affected by gender or menopausal status and there was no clear age effect.

PUFA synthesis was constitutive in the Jurkat T-lymphocyte leukaemic cell-line and was higher than in PBMCs. FADS2, FADS1 and ELOVL5 mRNA expression was also higher in Jurkat cells and was associated with 50% lower methylation of 17 CpGs in the FADS2 promoter, suggesting transcriptional dysregulation of PUFA synthesis in Jurkat cells involves altered DNA methylation.

These findings have provided novel insights into the regulation of PUFA biosynthesis in PBMCs and upregulation of the pathway in activated PBMCs suggests that newly synthesised PUFAs may be important for cell function.

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 (d5 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
( higher fat yogurts, nonetheless 55% (285 of 518 low-fat
yogurts) contained between 10 and 20 g sugar/100 g.
Within the children?s category, fromage frais had higher
protein (5.3 g/100 g (3.3, 8.6) vs 3.2 (2.8, 7.1); p and calcium contents (150 mg/100 g (90, 240) vs
130.5 mg/100 g (114, 258); p=0.0015) than yogurts.
Conclusions: While there is good evidence that yogurt
can be beneficial to health, products on the market vary
widely in total sugars. Fewer than 9%, and only 2% of
the children?s, products surveyed were low enough in
sugar to earn ?green? in UK front of the pack labelling.
Reformulation for the reduction of free sugars in yogurts is
warranted.

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, PÂ0.05). 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.

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 (rd0.35) 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.

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 ChREBP± impairs ChREBP± translocation into the nucleus and induction of ChREBP², 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.

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.

Background: Cardiovascular disease (CVD) remains a significant cause of mortality globally, with a reduction in free sugars consumption a public health target for decreasing disease burden. Fructose raises post-prandial triacylglycerol (TAG) concentrations (risk factor for CVD), relative to glucose or sucrose, possibly due disrupted intestinal TAG metabolism.
Aims: Determine whether effects of high-fructose consumption on plasma TAG concentration are explained by intestinal and/or hepatic TAG lipoprotein kinetics, as well as de novo lipogenesis (DNL), in humans and an in vitro model.
Methods: Five overweight males consumed drinks containing 30% energy as fructose or complex carbohydrate hourly for 11h on two separate occasions, in a randomised cross-over design (4 week washout). Stable isotopes were given to measure DNL, chylomicron (CM)-TAG and VLDL-TAG kinetics. CM and VLDL were separated according to their apolipoprotein B (apoB) content (Sf>20 fraction). Caco-2 cells were treated (96h) with different fructose and glucose concentrations, or a mixture of sugars (?Mix?), and stable isotopes tracers ([13C6]-fructose; [13C6]-glucose). TAG enrichments were measured by gas chromatography-mass spectrometry.
Results: Fructose consumption in comparison to the complex CHO had no effect on either intestinal or hepatic DNL. Intestinal and hepatic DNL were correlated in the fasted (P Conclusions: Fructose increased post-prandial TAG concentrations, possibly due to less insulin-mediated lipoprotein metabolism. DNL was unaffected by fructose consumption. This study supports public strategies to restrict dietary free sugars.

Fish skin, a by-product of filleting could be upgraded to value-added products for human consumption, thereby preventing waste and pollution. The aims were to prepare gelatin from skin of Nile or black tilapia (Oreochromis nilotica); characterize its physical-chemical, rheological and thermal properties and those of fish gelatin-whey protein mixtures; prepare hydrolysates and assess their foaming properties in the presence and absence of whey protein and green tea.
Gelatin extracted successfully from tilapia skins, gave a 19-26% yield and comprised high protein (90.2 + 0.68 %) and low fat (0.62 + 0.03 %), moisture (5.2 + 0.62 %) and ash content (0.08 + 0.05 %). Amino acid profile included mainly glycine (33.94%), alanine (26.1%) and glutamic acid, proline, arginine, aspartic acid and imino acids. The Bloom strength of 6.67% (w/v) tilapia gelatin in distilled water was higher (301 + 11.6 g) than commercial tilapia (207 + 5.6 g), bovine (~ 225 g), and porcine (~ 300 g) gelatins.
The rheological properties of 3,5 and 10% (w/v) tilapia gelatin indicated G? (elastic modulus) values 1,135 and 720 Pa, respectively, and gelling points of 16.2°C, 5% 17.2°C and 19.8°C respectively whereas melting temperatures were 14.9°C, 14.9°C and 15.0°C respectively. The gels were highly stable over a frequency range of 0-100 rad/sec.
DSC results indicated high melting temperatures (Tm) 22oC, compared to bovine (26.2°C) and porcine (30.7°C) gelatin. Cold water fish gelatin did not gel. However, bovine and porcine gelatins had lower enthalpy change (?H) values 0.58 J/g and 0.45 J/g respectively, compared with tilapia gelatin (2.51 J/g), probably due to protein denaturation of collagen.
In tilapia gelatin-whey protein mixed gels, whey proteins were dominant. Gelling temperatures of 10% WPI with 5% gelatin (88.9°C) or 10% gelatin (87.4°C) mixtures were higher than for WPI (80.3°C) or gelatin at 3% (16.2°C), 5% (17.2°C) and 10% (19.8°C). Whey protein (10%) was unstable but formed a good stable gel network with gelatin at 5 % (597 Pa) and 10% (26974 Pa). Whey (10 %) and gelatin (10%) also gave a strong gel by large deformation analysis. Tm and ?H for 10% whey protein and 5 or 10% gelatin/ mixtures were higher (67.2°C, 68.8°C and 68.2°C respectively) than for gelatin or whey protein alone. The ?H values of gelatins increased when combined with the WPI. Three % gelatin (0.03 J/g), 10% WPI + 3% gelatin (0.44 J/g), 5 % gelatin (0.18 J/g), 10% WPI + 5% gelatin (0.56), 10% gelatin (1.3 J/g) and 10% WPI + 10% gelatin (0.59). Phase contrast microscopy of gelatin showed a fine uniform and homogeneous network with small particles whereas the whey protein structure had larger aggregates. The gelatin-whey mixtures formed a compatible network.
Gelatin produced the highest volume of foam FE (933%), followed by gelatin + whey protein (853%), gelatin hydrolysate + whey protein (767%); these were not significantly different (p>0.05). This was followed by a mixture of gelatin + whey protein + green tea (575%) that was significantly lower (p