Jennifer Collins

Leukocyte Activation by Triglyceride-Rich Lipoproteins

Objective—Postprandial lipemia has been linked to atherosclerosis and inflammation. Because leukocyte activation is obligatory for atherogenesis, leukocyte activation by triglyceride-rich lipoproteins (TRLs) was investigated. Methods and Results—The expression of CD11b and CD66b after incubation with glucose and native and artificial TRLs (NTRL and ATRL) in vivo and in vitro was evaluated by flowcytometry. Oral fat loading tests showed an increased expression of CD11b on monocytes and neutrophils and CD66b on neutrophils. In 11 volunteers, postprandial leukocytes became enriched with meal-derived fatty acids ([1-13C]16:0) suggesting uptake of exogenous fat. ApoB binding on leukocytes measured by flowcytometry in 65 subjects was highest on neutrophils and monocytes suggesting adherence of apoB-containing lipoproteins. Physiological concentrations of TRLs showed 62% increased neutrophil CD11b and a dose-dependent increased monocyte CD11b up to 84% in vitro. Incubations with lipid emulsions in the hypertriglyceridemic range showed a 5-fold increased monocyte CD11b expression, which was higher than the positive control (fMLP), and a dose-dependent 2- to 3-fold increased neutrophil CD11b and CD66b. The oxidative scavenger DMTU decreased the neutrophil CD66b expression by 36%. Conclusion—Acute hypertriglyceridemia is a leukocyte activator most likely by direct interaction between TRLs and leukocytes and uptake of fatty acids. TG-mediated leukocyte activation is an alternative proinflammatory and proatherogenic mechanism of hypertriglyceridemia in part associated to the generation of oxidative stress.

De Novo Lipogenesis and Stearoyl-CoA Desaturase Are Coordinately Regulated in the Human Adipocyte and Protect against Palmitate-induced Cell Injury

De novo lipogenesis (DNL) is paradoxically up-regulated by its end product, saturated fatty acids (SAFAs). We tested the hypothesis that SAFA-induced up-regulation of DNL reflects coordinate up-regulation of elongation and desaturation pathways for disposal of SAFAs and production of monounsaturated fatty acids to protect cells from SAFA toxicity. Human preadipocytes were differentiated in vitro for 14 days with [U-13C]palmitate (0–200 μm) to distinguish exogenous fatty acids from those synthesized by DNL. Exogenous palmitate up-regulated DNL (p < 0.001) concomitantly with SCD and elongation (each p < 0.001). Adipocytes from some donors were intolerant to high palmitate concentrations (400 μm). Palmitate-intolerant cells showed lower TG accumulation. They had lower expression of SCD mRNA and less monounsaturated fatty acids in TG, emphasizing the importance of desaturation for dealing with exogenous SAFAs. There was greater [U-13C]palmitate incorporation in phospholipids. SCD knockdown with small interfering RNA caused down-regulation of DNL and of expression of DNL-related genes, with reduced membrane fluidity (p < 0.02) and insulin sensitivity (p < 0.01), compared with scrambled small interfering RNA controls. There was preferential channeling of DNL-derived versus exogenous palmitate into elongation and of DNL-derived versus exogenous stearate into desaturation. DNL may not act primarily to increase fat stores but may serve as a key regulator, in tandem with elongation and desaturation, to maintain cell membrane fluidity and insulin sensitivity within the human adipocyte.

De novo lipogenesis in the differentiating human adipocyte can provide all fatty acids necessary for maturation

The primary products of de novo lipogenesis (DNL) are saturated fatty acids, which confer adverse cellular effects. Human adipocytes differentiated with no exogenous fat accumulated triacylglycerol (TG) in lipid droplets and differentiated normally. TG composition showed the products of DNL (saturated fatty acids from 12:0 to 18:0) together with unsaturated fatty acids (particularly 16:1n-7 and 18:1n-9) produced by elongation/desaturation. There was parallel upregulation of expression of genes involved in DNL and in fatty acid elongation and desaturation, suggesting coordinated control of expression. Enzyme products (desaturation ratios, elongation ratios, and total pathway flux) were also correlated with mRNA levels. We used 13C-labeled substrates to study the pathway of DNL. Glucose (5 mM or 17.5 mM in the medium) provided less than half the carbon used for DNL (42% and 47%, respectively). Glutamine (2 mM) provided 9-10%, depending upon glucose concentration. In contrast, glucose provided most (72%) of the carbon of TG-glycerol. Pathway analysis using mass isotopomer distribution analysis (MIDA) revealed that the pathway for conversion of glucose to palmitate is complex. DNL in human fat cells is tightly coupled with further modification of fatty acids to produce a range of saturated and unsaturated fatty acids consistent with normal maturation.

Fraud by Abuse of Position and Unlicensed Gangmasters

This article analyses the Court of Appeal’s interpretation of the fraud by abuse of position offence in R v Valujevs. Two issues are explored: first, the Court’s welcome clarification of the meaning of a relevant ‘expectation’; second, the inadequacy of the Court’s reasoning in deciding that an unlicensed gangmaster ‘is expected to safeguard, or not to act against, the financial interests’ of his workers.

I2 Huntington’s disease reduced penetrance alleles occur at high frequency and affect age-related increases in prevalence

Background Huntington’s disease (HD) is diagnosed in 1 in 7300 individuals in Western populations but the frequency and penetrance of the causative CAG repeat expansion is unknown. Effects of population ageing, which may increase the rate of late-onset HD, remain unclear. Aims To directly estimate the frequency and penetrance of CAG repeat alleles associated with HD, and model changes in prevalence resulting from increased ascertainment of late-onset cases. Methods CAG repeat length was evaluated in 7315 individuals from three population-based cohorts in British Columbia, the United States, and Scotland. The frequency of CAG 36–38 repeat genotypes was compared to the prevalence of HD patients with genetically confirmed CAG 36–38 in a multisource clinical ascertainment in British Columbia, Canada. Penetrance of 36–38 CAG repeat alleles for HD was directly estimated for individuals ≥65 years of age. Age-specific prevalence rates were used to model change in prevalence as a function of population ageing and increased ascertainment of patients ≥65 years of age. Results 18 of 7315 individuals had ≥36 CAG, revealing that approximately 1 in 400 individuals in the general population have an expanded CAG repeat associated with HD (0.246%). Individuals with CAG 36–37 genotypes are the most common (36, 0.096%; 37, 0.082%; 38, 0.027%; 39, 0.000%; ≥40, 0.041%). The prevalence of HD is expected to increase as a result of both population ageing and increased ascertainment of late-onset cases. Conclusions The relatively infrequent diagnosis of HD at 36–38 CAG repeats suggests low penetrance in this range. Another contributing factor may be reduced ascertainment of HD in those of older age. Our data imply that population ageing will lead to higher prevalence rates of HD. Improved ascertainment of late-onset HD, particularly in the reduced penetrance range, may lead to further increases.

Abstract 1897: Metformin treatment leads to the accumulation of lipid in breast cancer cell lines - a potential new biomarker of response and mechanism of action.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC There is growing interest in the anticancer effect of the diabetes drug, metformin, and several clinical studies are underway worldwide. Metformins anticancer effects are thought to be mediated by its activation of AMPK and subsequent downstream catabolic effects on protein and lipid metabolism pathways that are commonly dysregulated in cancer cells. We have used mass spectrometry techniques and stable isotopes to investigate the effects of metformin on breast cancer cell lipid metabolism. There was great variability in lipid metabolism response to 2mM metformin treatment in a panel breast cancer cell lines (MCF7, BT474, ZR-75-1, T47D MDA-MB-157, MDA-MB-468 and MDA-MB-231). Levels of stearic acid (p=0.019), palmitoleic acid (p=0.01), oleic acid (p=0.002), cis-vaccenic acid (p=0.003) increased 3-4 fold with metformin treatment in MCF7 cells but there was no significant change for MDA-MB-231 cells. 2mM Metformin decreased fatty acid β-oxidation of oleate 4-fold (p=0.03). Accumulation of de novo lipogenesis derived (p=0.004) and exogenous fatty acid (p=0.02) occurred in MCF7 cells but siRNA knockdown of AMPK did not abrogate these effects. A 3-fold decrease in the diglyceride to triglyceride ratio (p=0.02) suggested reduced lipolysis and inhibited adipose triglyceride lipase activity. Reduced incorporation of exogenous fatty acid into phospholipid was evident (p=0.002). Heterogeneity of lipid metabolism response correlated with effects on proliferation, oxygen consumption and mitochondrial morphology. Our data shows that metformin has marked effects on accumulation of fatty acid in breast cancer cells most likely due to reduced lipolysis and β-oxidation and these effects cannot be explained as a result of AMPK activation. This work will contribute to developing biomarkers for metformins metabolic anticancer effect and our understanding of how to combine it with other therapies to exploit synthetic lethality. We have initiated a multicenter, phase II, single arm clinical study, to further characterise metformin influenced, cancer relevant metabolic pathways. We will look to assess the correlation between metabolic biomarkers, including the use of mass spectrometry and gas chromatography analysis of tissue samples, and metabolic response using 18F-FDG PET-CT scans. Citation Format: Simon Lord, Jennifer Collins, Barbara Fielding, Neel Patel, Fergus Gleeson, Adrian Harris, Fredrik Karpe. Metformin treatment leads to the accumulation of lipid in breast cancer cell lines - a potential new biomarker of response and mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1897. doi:10.1158/1538-7445.AM2013-1897