• Differential expression of Lp-PLA2 in obesity and type 2 diabetes and the influence of lipids

      Jackisch, L; Kumsaiyai, W; Moore, J.D; Al-Daghri, N; Kyrou, I; Barber, T.M; Randeva, H; Kumar, S; Tripathi, G; McTernan, P.G; et al. (Springer, 09/02/2018)
      Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a circulatory macrophage-derived factor that increases with obesity and leads to a higher risk of cardiovascular disease (CVD). Despite this, its role in adipose tissue and the adipocyte is unknown. Therefore, the aims of this study were to clarify the expression of Lp-PLA2 in relation to different adipose tissue depots and type 2 diabetes, and ascertain whether markers of obesity and type 2 diabetes correlate with circulating Lp-PLA2. A final aim was to evaluate the effect of cholesterol on cellular Lp-PLA2 in an in vitro adipocyte model. Analysis of anthropometric and biochemical variables from a cohort of lean (age 44.4 ± 6.2 years; BMI 22.15 ± 1.8 kg/m2, n = 23), overweight (age 45.4 ± 12.3 years; BMI 26.99 ± 1.5 kg/m2, n = 24), obese (age 49.0 ± 9.1 years; BMI 33.74 ± 3.3 kg/m2, n = 32) and type 2 diabetic women (age 53.0 ± 6.13 years; BMI 35.08 ± 8.6 kg/m2, n = 35), as part of an ethically approved study. Gene and protein expression of PLA2 and its isoforms were assessed in adipose tissue samples, with serum analysis undertaken to assess circulating Lp-PLA2 and its association with cardiometabolic risk markers. A human adipocyte cell model, Chub-S7, was used to address the intracellular change in Lp-PLA2 in adipocytes Lp-PLA2 and calcium-independent PLA2 (iPLA2) isoforms were altered by adiposity, as shown by microarray analysis (p < 0.05). Type 2 diabetes status was also observed to significantly alter gene and protein levels of Lp-PLA2 in abdominal subcutaneous (AbdSc) (p < 0.01), but not omental, adipose tissue. Furthermore, multivariate stepwise regression analysis of circulating Lp-PLA2 and metabolic markers revealed that the greatest predictor of Lp-PLA2 in non-diabetic individuals was LDL-cholesterol (p = 0.004). Additionally, in people with type 2 diabetes, oxidised LDL (oxLDL), triacylglycerols and HDL-cholesterol appeared important predictors, accounting for 59.7% of the variance (p < 0.001). Subsequent in vitro studies determined human adipocytes to be a source of Lp-PLA2, as confirmed by mRNA expression, protein levels and immunochemistry. Further in vitro experiments revealed that treatment with LDL-cholesterol or oxLDL resulted in significant upregulation of Lp-PLA2, while inhibition of Lp-PLA2 reduced oxLDL production by 19.8% (p < 0.05). Our study suggests adipose tissue and adipocytes are active sources of Lp-PLA2, with differential regulation by fat depot and metabolic state. Moreover, levels of circulating Lp-PLA2 appear to be influenced by unfavourable lipid profiles in type 2 diabetes, which may occur in part through regulation of LDL-cholesterol and oxLDL metabolism in adipocytes.
    • Tunicamycin-induced ER stress mediates mitochondrial dysfunction in human adipocytes

      Jackisch, L; Murphy, A; Al-Daghri, N; McTernan, P; Randeva, H; Tripathi, G; University of Westminster (bioscientifica, 01/03/2014)
      The pathogenesis of obesity and T2DM mediates mitochondrial dysfunction which, in part, may arise as a consequence of endoplasmic reticulum (ER) stress. However, the potential impact of ER stress on mitochondria dysfunction is unclear. Therefore, we investigated whether induction of ER stress contributes to mitochondrial dysfunction in human adipocytes using 1) human differentiated adipocyte cell line (Chub-S7, n=12); and 2) primary differentiated lean and obese abdominal subcutaneous adipocytes (AbdSc Ad; n=3 respectively). ER stress was induced in post-differentiated Chub-S7 (AbdSc Ad) using tunicamycin (Tn) (0.25 μg/ml, 0.75 μg/ml) for 24 hrs, 48 hrs and 72 hrs. Assessment of mitochondrial function post Tn treatment was undertaken using the Extracellular Flux Analyser – evaluating oxygen consumption rate (OCR) and proton excretion (glycolysis; extracellular acidification rates (ECAR)). Flux stressors (oligomycin, FCCP, rotenone/antimycin A) were given to Chub-S7 adipocytes treated with Tn to measure mitochondrial response. Mitochondrial dynamics were also evaluated using RT-PCR and confocal microscopy. The Seahorse stress test identified that Tn (0.25 μg/ml, 0.75 μg/ml) induced mitochondrial stress with a 14% rise in OCR (Basal: 472 pMoles/min vs Tn: 537 pMoles/min; P=0.002) and a maximum 78% increase in ECAR (Basal: 124 mpH/minute vs Tn: 228 mpH/minute; P=0.006). This Tn induced mitochondrial stress was maintained over 72 hrs. Coupled with the observed functional data, mRNA expression analysis highlighted that fission (Drp1, Fis 1; P<0.01) and fusion (Mfn2, Opa1; P<0.01) were both increased by Tn (0.25 μg/ml, 0.75 μg/ml). Confocal microscopy was used to further verify this result. These studies highlight unfavourable changes in mitochondrial function and gene expression arise in adipocytes, in response to an inducer of ER stress; this may mimick an obese phenotype. Taken together, these results indicate that therapeutics to reduce ER stress could have a beneficial influence on alleviating mitochondrial dysfunction and its pathogenic consequences.