• Accelerometer-based physical activity levels differ between week and weekend ways in British preschool children

      Roscoe, Clare M. P.; James, Rob S.; Duncan, Michael J.; University of Derby; Coventry University (MDPI AG, 2019-09-12)
      Participation in physical activity (PA) is fundamental to children’s future health. Studies examining the temporal pattern of PA between weekdays and weekends in British preschool children are lacking. Therefore, the aim of this study was to compare PA levels between week and weekend days for UK preschool children, using objective measurements. One hundred and eighty-five preschool children (99 boys, 86 girls, aged 4–5 years), from central England wore a triaxial accelerometer (GENEActiv) for 4 days to determine PA. The time (min) and percentage (%) of time spent in light, moderate and vigorous PA (MVPA) was determined using specific cut-points for counts per minute related to 3–5 year olds. Of the sample, none of the children met the UK recommended 180 min or more of PA per day. A significant difference (P < 0.05) was observed between the amount of time that preschool children spent in sedentary behaviours on weekdays (91.9%) compared to weekend days (96.9%). During weekdays and weekend days, 6.3% and 2.0% of time was spent in MVPA, respectively. Therefore, a substantial proportion of British preschool children’s day is spent in sedentary behaviours, with less MVPA accrued during the weekend. Regular engagement during the weekdays provides opportunities to accrue PA, which may not be present on weekend days.
    • Do individuals with Chronic Pain show attentional bias to pain-related information? An early stage systematic review of the eye-tracking evidence.

      Gaffiero, Daniel; Elander, James; Maratos, Frances; University of Derby (British Psychology Society, 2019-03)
    • Treating hoarding disorder with compassion‐focused therapy: A pilot study examining treatment feasibility, acceptability, and exploring treatment effects

      Chou, Chia‐Ying; Tsoh, Janice Y.; Shumway, Martha; Smith, Lauren C.; Chan, Joanne; Delucchi, Kevin; Tirch, Dennis; Gilbert, Paul; Mathews, Carol A.; Department of Psychiatry, University of California, San Francisco, California, USA; et al. (Wiley Online Library, 2019-07-04)
      Hoarding disorder (HD) was recognized as a psychiatric disorder in 2013. Existing literature suggests room for improvement in its treatment. The current pilot study aimed to provide an initial evaluation on the potential of compassion‐focused therapy (CFT) as an intervention for HD, with the primary aim being assessing its feasibility and acceptability, and the secondary being evaluating its effects. Both CFT and a second round of the current standard of treatment and cognitive behavioural therapy (CBT) were investigated in the current study as follow‐up treatment options for individuals who had completed CBT but were still significantly symptomatic. Forty eligible individuals were enrolled (20 in each treatment). Treatment feasibility and acceptability were assessed by quantitative and qualitative measures. To explore treatment effects, HD symptom severity, HD‐related dysfunctions, and their underlying mechanisms were assessed pre‐treatment and post‐treatment. Retention rates were 72% for CFT and 37% for CBT. All participants and 79% of the participants rated CFT and CBT, respectively, as good or excellent. After receiving CFT as a follow‐up treatment, HD symptom severity dropped below the cut‐off point for clinically significant HD for 77% of the treatment completers, and 62% achieved clinically significant reduction in symptom severity. In contrast, after completing a second course of CBT, 23% had HD symptom severity dropped below the cut‐off threshold, and 29% achieved clinically significant symptom reduction. The current study showed satisfactory feasibility and acceptability of CFT. Moreover, it also found promising effects of CFT in addressing hoarding‐related mechanisms that may not have been sufficiently addressed by CBT. The results suggest promising potential of CFT as a treatment for HD. Further investigation on this intervention is needed. CFT may be a promising treatment option, particularly for those who do not respond well to CBT. Improving emotion regulation and negative self‐perception by applying CFT interventions may help relieve hoarding symptoms. Generalization of the findings should be applied with caution given the small convenience sample of the current study. Statistical comparison on treatment effect measures between CFT and CBT as follow‐up treatments was not available due to small sample size. Therefore, the comparative conclusions based on this pilot study should be made with caution.
    • PWE-001 field cancerisation theory in colorectal cancer (crc): what role do fibroblast growth factors have?

      Patel, A; Williams, N; Nwokolo, C; Tripathi, G; Arasaradnam, R; University of Westminster (BMJ, 09/06/2014)
      Characterisation of the molecular field defect around colorectal cancer (CRC) could enable identification of novel biomarkers that could be used for early detection of CRC. Previous studies have suggested fibroblast growth factor 19 (FGF19) may play a role in CRC formation through interaction with the B-catenin/wnt signalling cascade. The role of fibroblast growth factor 7 (FGF7) however remains controversial. The aim of this study was to determine if there are differences in FGF19 and FGF7 gene expression in cancer tissue and the adjacent ‘normal tissue’ compared with normal colonic tissue. Mucosal pinch biopsies were taken from the rectum and caecum at time of colonoscopy for healthy controls. For CRC patients, tissue samples were taken from the tumour, adjacent to the tumour and at the resection margin of the colectomy specimen. Healthy controls were age and sex matched to CRC patients. Quantitative real time PCR was used to determine gene expression of FGF19, its receptor FGFR4, FGF7 and its receptor, FGFR2. Results were further validated using immunohistochemistry. Serum levels of FGF19 were measured using the Quantikine ELISA kit (RandD systems, UK). 49 patients were recruited (28 M: 21 F, median age 71 years (range 48–86 years)); 18 patients with CRC and 32 healthy controls. There was no overall difference in gene expression of FGF19/FGFR4 or FGF7/FGFR2 between cancer patients and healthy controls. There was upregulation of FGFR4 in mucosa adjacent to the tumour (mean fold change 1.23 vs. 0.93, p = 0.38) and the tumour itself (mean fold change 1.49 vs. 1.04, p = 0.700) in patients whose tumour expressed FGF19 compared to those that did not. Patients with upregulation of FGF19/FGFR4 had a significantly lower fasting serum FGF19 level (119 pg/ml versus 208 pg/ml, p = 0.05). FGF7 was upregulated in 6/19 cancers; this was associated with a significant upregulation in FGF7 in adjacent mucosa compared with cancers where FGF7 was downregulated (mean fold change 3.62 vs. 0.95, p = 0.018). There was a non-significant trend towards upregulation of the receptor (FGFR2) in mucosa adjacent to the cancer and the tumour tissue itself. Upregulation of FGFR4 in patients whose tumours expressed FGF19 corresponded inversely with serum FGF19 suggesting its potential as a putative biomarker. Significant upregulation of FGF7 in ‘normal’ mucosa adjacent to only tumours that express FGF7 lends support to the field theory of colorectal carcinogenesis.
    • Systemic triglycerides as a key determinant of TLR regulated inflammatory risk in human adipose tissue post bariatric surgical intervention and weight loss

      Kumsaiyai, W; Al-Daghri, N; Kyrou, I; Vrbikova, J; Hainer, V; Fried, M; Sramkova, P; Barber, T; S Kumar; Tripathi, G; et al. (bioscientifica, 01/03/2014)
      Bariatric surgery can lead to a quick reversal in type 2 diabetes mellitus (T2DM) status. However, despite this reversal inflammatory responses may still persist via activation of Toll-like receptors (TLR) within adipose tissue (AT); with triglycerides (TGs) noted as a potential mediator of such inflammation. Therefore the aims of these studies were to understand the impact of TG changes, pre- and post-bariatric surgery, on TLR expression in ex vivo AT and the in vitro effects of triglyceride rich lipoprotein (VLDL), on TLR expression in isolated human differentiated pre-adipocytes. Obese, T2DM, female subjects (age: 54.6±6.6 years, BMI pre (41.2±5.5 kg/m2) and 6 months post-surgery (36.05±5.16 kg/m2; n=30) underwent bariatric surgery (banding (n=8); plication (n=14); and biliopancreatic diversion (n=8)). Biochemical data and abdominal subcutaneous AT (AbdSc AT) samples were taken during surgery and 6 months post-surgery. Real-time PCR assessed TLR expression. Human differentiated pre-adipocyte Chub S7 cells were used to examine transcriptional effects of VLDL on TLR expression. Following surgical intervention, BMI (P<0.001), blood glucose (P<0.001), insulin (P<0.001), HOMA-IR (P<0.001), TG (P<0.05), cholesterol (P<0.001), and LDL-cholesterol (P<0.05) were significantly improved. There was a significant reduction in TLR4 mRNA post-surgery (P<0.01) irrespective of surgery type. It was also noted that subjects with the greatest drop (55.5% reduction) in TGs post-surgery (P<0.001) showed a significant correlated reduction in TLR4 mRNA expression (P<0.001). In vitro treatment of differentiated Chub S7 cells highlighted VLDL induced TLR4 mRNA expression (P<0.05).There is a reduction in AT inflammation as denoted by TLR expression. The reduction in AT inflammation appears dependent on how successfully subjects reduce their serum triglyceride, which is supported by in vitro studies. These studies suggest that bariatric surgery lead to metabolic improvement with weight loss, whilst dietary intervention is still required to ensure TGs reduce to reduce inflammation.
    • Meal size and frequency influences metabolic endotoxaemia and inflammatory risk but has no effect on diet induced thermogenesis in either lean or obese subjects

      Piya, M; Reddy, N; Campbell, A; Hattersley, J; Halder, L; Tripathi, G; Tahrani, A; Kumar, S; Barber, T; McTernan, P; et al. (bioscientifica, 01/03/2014)
      Small frequent meals are often recommended for weight loss, with supporting evidence often provided from studies in diabetes. Dietary meal content is also relevant, as high fat meals cause systemic inflammation via gut derived bacteria, endotoxin. As such, repeated meals may exacerbate this. In contrast, dietary induced thermogenesis, related to meal size, may reduce with small frequent meals. Therefore, the aim of this study was to compare the effect of 2 vs 5 meals on metabolic endotoxaemia and 24 h (hour) energy expenditure in lean and obese women. In a crossover study, 24 lean (age: 34 (mean±S.D.)±10 years, BMI: 22.9±2 kg/m2) and obese (age: 42±9 years, BMI: 36±8 kg/m2) women were given two or five isocaloric high (50%) fat meals, on two separate days. On both visits, 24 h energy expenditure was measured in whole body room calorimeters and blood samples taken 2 hourly (0900 to 2100 h). Serum endotoxin, glucose, insulin, lipids were measured. The obese subjects had increased area under the curve (AUC) for insulin, glucose, HOMA-IR and triglyceride (TG), with decreased HDL (P<0.01), compared with lean subjects, for both meal visits. For the entire cohort, fasting endotoxin correlated with triglyceride (r=0.32, P<0.05), and AUC for endotoxin and TG correlated in the five meal visit (r=0.44, P<0.05), but not the two meal visit. In the final 2100 h blood test, the endotoxin levels were significantly higher in the five meal visit (P=0.05), but not the two meal visit. Meal frequency did not affect 24 h expenditure, in either the obese group (2124±312 vs 2142±365 Kcal/day) or lean group (1724±160 vs 1683±166 Kcal/day).Our findings suggest in metabolically healthy lean and obese subjects, increased meal frequency may pose an inflammatory risk posed by circulating endotoxin and TGs leading to peak levels at bedtime. As such, small frequent meals may not influence diet induced thermogenesis, but may increase metabolic disease risk.
    • Unfolded protein response in adipose tissue of obese diabetic women significantly improved 6 months post bariatric surgery, irrespective of malabsorptive or bypass operation type and correlates with plasma glucose concentration

      Voyias, P; Antonysunil, A; Kumasaiyai, W; Kyrou, I; Vrbikova, J; Hainer, V; Fried, M; Sramkova, P; Saravanan, P; Kumar, S; et al. (bioscientifica, 01/03/2014)
      In obesity, excess nutrients and an increased demand for protein synthesis contribute to unfolded proteins accumulating within the endoplasmic reticulum and consequent activation of unfolded protein response (UPR). UPR in adipose tissue (AT) is critical to the initiation and integration of inflammation and insulin signalling pathways in obese and type 2 diabetes mellitus (T2DM) patients. The aim of this study was to examine whether novel malabsorptive or bypass bariatric surgery in obese women with T2DM leads to reduction in UPR. Abdominal subcutaneous (AbSc) AT was isolated from 30 Caucasian obese T2DM women aged 54.1±1.3 (mean±S.E.M.) years, BMI 41.21±1.0 kg/m2, that had undergone bariatric surgery of malabsorptive; gastric band (n=9) or novel gastric plication (n=13), or bypass; biliopancreatic diversion (n=8) type. Biopsies and anthropometric data were collected at the time of surgery and 6 months post-surgery. UPR markers were measured by qRT-PCR and western blotting and correlation analysis was performed. Six months post-operation all subjects significantly reduced body weight (P<0.001) with mean excess BMI lost 33.4±2.4%. Anthropometric measurements were significantly improved; fat mass, HbA1c, glucose, insulin, HOMA-IR, and total cholesterol (all P<0.001). ATF6, IRE1α, XBP1s, ATF4, and CHOP10 mRNAs and ATF6, pIRE1α, XBP1s, Calnexin and Bip proteins were all significantly (P<0.05) reduced post-surgery irrespective of operation type. Correlations between UPR mRNAs were strengthened post-surgery for ATF4 and CHOP10 (P=0.041–P<0.001) and IRE1α and ATF6 (P=0.853–P<0.001). Post-surgery plasma glucose correlated significantly (P=0.034) with XBP1s mRNA. This study highlights that bariatric surgery induced weight loss is coupled with improved glucose homeostasis and reduced UPR expression in AT. Furthermore post weight loss there are enhanced associations identified between UPR and XBP1 in AT and plasma glucose which may arise due to improved glucose homeostasis. This suggests UPR regulation in AT is linked to plasma glucose levels which aligns to metabolic health.
    • A 3-month low fat diet leads to significant lipid profile improvement in obese T2DM Saudi subjects, without substantial weight loss, and the capacity to manage a damaging high-fat meal challenge more appropriately post intervention

      Al-Disi, D; Al-Daghri, N; Khan, N; Alsaif, M; Alfadda, A; Sabico, S; Tripathi, G; McTernan, P; University of Westminster (bioscientifica, 01/03/2014)
      Current evidence highlights that dietary cholesterol, trans-fatty acids and saturated fatty acids (SFAs) are all known to increase the levels of systemic atherogenic lipoproteins and cardiovascular disease. The aim of this study was to observe the direct effect of dietary change, via a calorie-restricted diet on i) cardio-metabolic profile and ii) a high-fat meal challenge pre- and post-3-month intervention. T2DM subjects (Saudi female, age: 40.50±6.8years, BMI: 37.28±10.75 kg/m2, n=18) were given a high-fat meal pre- and post-calorie restricted diet (3 months; 500 kcal deficit/day, balanced diet with complex carbohydrate). Baseline (0 h) and post-prandial sera (1–4 h) were taken from subjects, anthropometric and biochemical data was collated at both time points. On baseline comparison of pre- and post-diet interventions, there were modest reductions in anthropometric data, BMI (P<0.001), waist (P<0.001), and waist:hip ratio (WHR; P<0.01). Baseline HDL-cholesterol increased significantly (P<0.01) whilst LDL- and total-cholesterol were significantly reduced (pre-total cholesterol: 5.13 (4.53, 5.93) vs post-total cholesterol: 4.70 (4.01, 5.14); pre-LDL cholesterol: 3.56 (3.07, 4.06) vs post-LDL cholesterol: 2.81 (2.34, 3.56), P<0.05). The findings also showed significant changes in the effects of high-fat meal intake on the metabolic profile pre- and post-diet intervention. At 4 h post-prandially, post-dietary intervention, HDL-cholesterol was 16.6% higher than pre-diet (P<0.05), whilst LDL- and total-cholesterol were 24.2 and 12% lower, respectively, than at the 4 h equivalent pre-diet (P<0.05). These findings suggest that lipid mediators associated with increased cardiometabolic risk can be quickly reversed as a result of a balanced diet, in T2DM subjects without substantial weight loss. As a result, the body is able to cope with the occasional high-fat meal insult, whilst still maintaining a reduced long-term CVD risk. As such, this is a diet that patients with T2DM may be able to adhere to more successfully, longer-term.
    • Vitamin B12 deficiency induces cholesterol biosynthesis by limiting S-adenosyl methionine and altering the methylation of Srebf1 and Ldlr genes

      Adaikalakoteswari, A; Finer, S; Voyias, P.D; McCarthy, C; Moore, J; Smart-Halajko, M; Bawazeer, N; Al-Daghri, N.M; McTernan, P.G; University of Westminster
      Maternal vitamin B12 deficiency affecting one-carbonmetabolism influences metabolic status and the degree of insulinresistance of the offspring in adulthood. But its significance andmechanism in the development of adiposity and adipose tissuedysfunction is unknown. To investigate the role of vitamin B12 in the developmentof adipocyte dysfunction. Human pre-adipocytes were differentiatedin customised media with varying concentrations of B12. Adipo-cytes cultured in low B12 (0.15nM) or no B12 conditions hadincreased cholesterol and homocysteine levels and reduced glucoseuptake capacity compared to control (B12 500nM). Global DNAmethylation profiling and bisulphite pyrosequencing showed thatthe promoter regions of sterol regulatory element-binding tran-scription factor 1 (SREBF1) and low density lipoprotein receptor(LDLR) were hypomethylated in B12 deficient conditions, consis-tent with the increased gene expressions. The S-adenosyl methio-nine/S-adenosyl homocysteine ratio was significantly lower in B12deficient conditions. Inhibition of methylation in high B12 condi-tions by 5-aza-2-deoxycytidine led to increased cholesterol accu-mulation but not homocysteine. In two independent clinicalstudies, women at child bearing age (age: 19–39 years) and inearly pregnancy (16–18 weeks), showed that low B12 was asso-ciated with higher total cholesterol, LDL cholesterol and choles-terol to HDL ratio. Regression analysis in the pregnant cohortadjusting for all confounders showed B12 levels to be indepen-dently associated with total cholesterol and triglyceride levels. Vitamin B12 deficiency leads to adipocyte dysfunc-tion by inducing cholesterol biosynthesis and homocysteine.Induction of cholesterol biosynthesis was due to hypomethylationof SREBF1 and LDLR. Clinical observations support that the B12effect is independent and our findings show this link is probably causal.
    • Elevated cord leptin from low B12 mothers predicts birth weight

      Antonysunil, A; Vatish, M; Lawson, A; Wood, C; Sivakumar, K; Webster, C; Anderson, N; McTernan, P; Tripathi, G; Saravanan, P; et al. (bioscientifica, 01/03/2014)
      Vitamin B12 (B12) insufficiency is common in pregnancy and independently predicts insulin resistance (IR) in the offspring. B12 is an important key nutrient for epigenetic programming through regulating DNA methylation. Such B12 DNA methylation may influence leptin, a strong candidate for methylation, which could impact both insulin resistance (IR) and associated neonatal metabolic risk. Therefore, we hypothesize that leptin can be programmed by maternal B12 which could influence metabolic risk in the offspring. To test this hypothesis, we investigated whether i) maternal B12 is associated with leptin in cord blood and ii) evaluated their association with birth weight. Paired maternal venous and cord blood samples (n=91) were collected at the time of elective caesarean section. Serum vitamin-B12 was determined by electro-chemiluminescent immunoassay. Leptin levels were measured by ELISA. B12 insufficiency (<150 pmol/l) was common (mothers-40%; and neonates-29%). Maternal B12 was inversely associated with neonatal leptin (r=−0.304; P=0.005). In regression analysis, adjusted for all likely confounders, maternal B12 independently predicted neonatal leptin (β=−0.647; P=0.005; R2=12.8%). There was no correlation between maternal and neonatal leptin levels. Cord leptin from mothers with low B12 correlated with birth weight (r=0.366; P=0.036). Regression analysis adjusted for maternal leptin and insulin showed that cord leptin from mothers with low B12 independently predicted birth weight (β=0.024; P=0.049; R2=14.5%). Our study highlights that maternal B12 insufficiency predicts elevated leptin in cord blood and is associated with higher birth weight. Since cord leptin is derived from neonatal adipose tissue and not mother, these findings suggest that maternal B12 might program leptin levels in-utero either directly through the satiety centre or mediated via inducing IR and adiposity in the offspring. Delineating the mechanistic relationship between cord leptin and maternal B12 might provide crucial answers in understanding the molecular mechanisms of adverse metabolic programming.
    • Freeze dried broccoli extract relieves ER stress and mitochondrial inefficiency in differentiated human pre-adipocyte cells

      Murphy, A; Jackisch, L.; Azharian, S; Aladel, A; Barker, G; Tripathi, G; Chappell, M; McTernan, P; University of Westminster (bioscientifica, 01/03/2014)
      In obesity, excess nutrients can disrupt protein folding in the endoplasmic reticulum (ER) which activates the unfolded protein response (UPR) and alters mitochondrial function. These changes can induce inflammation, oxidative stress and insulin resistance. The aim of the study was to investigate whether broccoli extract can protect against cellular damage in human adipocytes, which with mathematical modelling may help predict pathway response. Differentiated Chub-S7 cells were treated over a 72 hr time course with 10 ng/ml freeze-dried broccoli extract (hybrid Brassica oleracea var. italic) alone or combined with ER stress inducer, tunicamycin (750 ng/ml). UPR markers (ATF6, ATF4, CHOP, ERO1α, P-PERK, PERK, P-eIF2α, eIF2α, P-IRE1α and IRE1α) were measured by qRT-PCR and Western blot. Mitochondrial genes (MFN2, OPA1, UCP2, SOD2, POLG) were also measured. Mathematical modelling was undertaken. Tunicamycin led to a significant increase in UPR gene expression (P<0.05), whilst broccoli extract combined with tunicamycin significantly reduced the expression of UPR markers compared with those treated only with tunicamycin, in a time dependent manner. Tunicamycin had a detrimental effect on mitochondrial genes (P<0.05); the presence of broccoli appeared to protect against these effects. This in-vitro time-series data are being used to realistically parameterise an existing mathematical model. Broccoli extract appears to positively influence protein folding in ER stressed adipocytes, reducing UPR gene expression and causing influential changes in mitochondria. As such broccoli supplementation in the daily diet may reduce the inflammatory response posed by adipose tissue during weight gain. The mathematical model of the UPR offers the possibility of in silico optimisation for the supplementation.
    • PWE-254 Is the macroscopically normal mucosa (MNM) around colorectal cancer really ‘normal’?

      Patel, A; Fang, Y; Moore, J; Williams, N; Tripathi, G; Arasaradnam, R; University of Westminster (BMJ, 22/06/2015)
      Field cancerisation refers to the process whereby cells acquire pro-tumourigenic mutations that predispose to malignant transformation but do not produce morphological change.1Previous colorectal cancer studies have assumed that the macroscopically normal mucosa (MNM) adjacent to a cancer is biologically unaltered. The aim of this study was to determine if the genetic expression profile of the MNM around a cancer or adenoma is different to that found in healthy controls. 15 patients undergoing colonoscopy were recruited over 12 months; 5 healthy controls, 5 with colorectal adenomas and 5 with adenocarcinoma. Two mucosal pinch biopsies were taken in the rectum, right colon and adjacent to polyp or cancer. mRNA was extracted and gene expression was assessed using standard whole genome micro-array analysis. Differentially expressed genes were identified using three methods of analysis: LIMMA (fold change ratio >1.5 and p value <0.05), Robust Regression (RR) (adjusted p value <0.05) and genes that ‘overlap’ when LIMMA (p value <0.001) and RR (adjusted p value <0.1) are used. Functional analysis was performed using DAVID2software to identify important biological processes that were dysregulated. A large number of genes were dysregulated in the MNM adjacent to cancer or adenoma compared with controls (Table 1). Interestingly, the greatest differences were seen between MNM adjacent to cancer and polyp in chromatin organisation, nucleosome processing, nuclear transport and histone assembly. The most significantly upregulated genes consisted of FUT2, CTSA, MUC2 and SDS and downregulated genes consisted of GREM1, SFRP, HIST1H, IL17B and TFF1.
    • 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.
    • Telmisartan reverses antiretroviral-induced adipocyte toxicity and insulin resistance in vitro

      Pushpakom, S.P; Adaikalakoteswari, A; Owen, A; Back, D.J; Tripathi, G; Kumar, S; McTernan, P; Pirmohamed, M; University of Warwick (Sage, 21/02/2018)
      Antiretroviral therapy in HIV-positive patients leads to insulin resistance which is central to the pathogenesis of various metabolic abnormalities and cardiovascular disease seen in this patient group. We have investigated the dose–response relationship of telmisartan, an antihypertensive, on adipocytes in vitro in order to determine whether it may have metabolic beneficial effects. Using in vitro chronic toxicity models (3T3-F442A murine and primary human adipocytes), we evaluated the effects of different concentrations of telmisartan on adipocyte differentiation and adipogenic gene expression using lipid accumulation assays and real-time polymerase chain reaction, respectively. Adipokine secretion and expression of insulin signalling mediators were evaluated using enzyme-linked immunosorbent assays. Telmisartan partially reversed the deleterious effects of antiretrovirals on adipocyte lipid accumulation, expression of adipogenic regulators (peroxisome proliferator receptor-gamma and lipin 1), adipokine secretion and expression of the insulin signalling mediator pAktSer473. The metabolic effects of telmisartan followed a non-monotonic response with the maximal effect observed at 5 µM in the primary human adipocyte model. Telmisartan has beneficial metabolic effects in adipocytes in vitro, but its potential to reduce antiretroviral-induced cardiometabolic disease in HIV-infected individuals needs to be evaluated in a well-designed adequately powered clinical trial.
    • Habitual physical activity is associated with circulating irisin in healthy controls but not in subjects with diabetes mellitus type 2

      Al‐Daghri, N.M; Alokail, M.S; Rahman, S; Amer, O.E; Al‐Attas, O.S; Alfawaz, H; Tripathi, G; Sabico, S.; Chrousos, G.P; McTernan, P.G; et al. (Wiley, 22/05/2015)
      Irisin, a novel myokine, has been shown to increase following vigorous exercise, with studies suggesting that it mediates some of the beneficial effects of exercise. Irisin might play a role in ‘browning’ of white adipocytes, thus increasing energy expenditure. The role of irisin in exercise and energy expenditure in subjects with diabetes mellitus type 2 (DMT2) remains largely unknown. We aimed to investigate the association between circulating irisin and habitual physical activity in subjects with and without DMT2. In this cross‐sectional study, 164 Saudi adults: 81 non‐DMT2 controls [age: (mean ± SD) 51·6 ± 10·9; BMI: 29·6 ± 4·3 kg/m2] and 83 DMT2 subjects [age: 54·3 ± 10·3 year; BMI: 29·4 ± 4·7 kg/m2] were studied. Anthropometric and fasting serum biochemical data were collected. Circulating irisin was measured using an enzyme‐linked immunosorbent assay (ELISA). Frequency intensity time (FIT) index was used to assess the level of habitual physical activity. We observed significantly higher levels of irisin in DMT2 subjects than in controls (P < 0·001). FIT index was positively associated (r = 0·20, P = 0·03) with circulating irisin in controls only. Additionally, irisin levels were significantly higher in tertile 3 (0·75 ± 0·07 μg/mL) than tertile 1 (0·49 ± 0·06 μg/mL) of the FIT index in healthy controls, whilst no such relation with physical activity was observed in DMT2 subjects. This cross‐sectional study has shown a weak association of irisin with physical activity levels in healthy controls but not in DMT2 subjects, suggesting the possibility of discordant regulation in the condition of DMT2.
    • Novel splicing variants of recepteur d'origine nantais (RON) tyrosine kinase involving exons 15–19 in lung cancer

      Krishnaswamy, S; Mohammed, A.K; Amer, O.E; Tripathi, G; Alokail, M.S; Al-Daghri, N.M; University of Westminster (Elsevier, 12/12/2015)
      Altered expressions of receptor tyrosine kinases drive the growth and metastasis of several cancers. RON is a single pass transmembrane receptor tyrosine kinase (RTK) shown to be aberrantly expressed in various cancer types. However, target validation and successful therapeutic targeting of RON in cancers is hampered by the co-existence of unknown number/types of isoforms, which are structurally similar but functionally diverse. The objective of this study was to identify differential splicing in the C-terminal region of RON transcripts to better understand RON signaling in cancers. mRNA transcript sequence between exons 14 and 20 of RON was PCR amplified and sequenced using cDNA from 10 SCLC and 13 NSCLC cell lines. Specific exon deletions were identified by aligning sequencing chromatograms with reference RON cDNA sequence. We identified the presence of four unique transcript sequence variants of RON formed through skipping of exons 15–19, 16–19, 16–17 and 16. The transcript variants, except the one lacking exons 15–19, were found in more than one cell line. Several cell lines contained two to four of these uniquely spliced transcript variants. dbEST (Expressed Sequence Tags database) or other DNA sequence databases did not contain RON cDNA sequences corresponding to any of the above exon deletions indicating that all these transcript sequence alterations are novel. Results of our study indicate common occurrence of different types of alternatively spliced transcripts of RON in lung cancer with potential to be translated into proteins lacking active kinase domain. Our findings suggest that tumors produce several dominant negative isoforms which probably inhibit ligand dependent RON signaling, and hence, raise important questions regarding the appropriateness of blocking wild type RON signaling for therapy. Further, presence of transcript variants and their isoform products may interfere with quantitative and functional analysis during target validation.
    • 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.
    • Identification of the splice variants of Recepteur d'Origine nantais (RON) in lung cancer cell lines

      Krishnaswamy, S; Bukhari, I; Mohammed, A.K; Amer, O.E; Tripathi, G; Alokail, M.S; Al-Daghri, N.M; University of Westminster (Elsevier, 14/09/2018)
      RON receptor tyrosine kinase is a transmembrane protein directly involved in suppression of inflammation and its aberrant expression linked to cancers and metastasis. Efforts to block deregulated RON signaling in tumors using small molecule kinase inhibitors or antibodies have been complicated by the presence of unknown number/types of isoforms of RON, which, despite being structurally similar, localize differently and mediate varied functions. Current study was designed to identify the splice variants of RON transcripts formed by skipping of sequences between exons 9 and 14 for better understanding of isoform specific RON signaling in cancers. PCR amplification and bi-directional sequencing of a 901 bp cDNA sequence located between exons 9 to 14 of RON from lung cancer cell lines revealed the presence of two splicing variants formed by skipping of exons 11 and 11–13. Each of these transcripts was found in more than one cell line. Expressed sequence tag (EST) database search indicated that the splicing variant lacking exons 11–13 was a novel one. Here we conclude that the splice variants of RON lacking exon 11 and exons 11–13 were detected in several lung cancer cell lines. Novel variant formed by skipping exons 11–13, the sequence of which code for transmembrane region, is predicted to code for a truncated isoform that may be secreted out. Tumors may antagonize the ligand dependent anti-inflammatory function of wild-type RON by secreting out the ligand binding isoforms.
    • Field cancerisation in colorectal cancer: A new frontier or pastures past?

      Patel, A; Tripathi, G; Gopalakrishnan, K; Williams, N; Arasaradnam, R.P; University of Westminster (Baishideng Publishing Group, 07/04/2015)
      Despite considerable advances in our understanding of cancer biology, early diagnosis of colorectal cancer remains elusive. Based on the adenoma-carcinoma sequence, cancer develops through the progressive accumulation of mutations in key genes that regulate cell growth. However, recent mathematical modelling suggests that some of these genetic events occur prior to the development of any discernible histological abnormality. Cells acquire pro-tumourigenic mutations that are not able to produce morphological change but predispose to cancer formation. These cells can grow to form large patches of mucosa from which a cancer arises. This process has been termed “field cancerisation”. It has received little attention in the scientific literature until recently. Several studies have now demonstrated cellular, genetic and epigenetic alterations in the macroscopically normal mucosa of colorectal cancer patients. In some reports, these changes were effectively utilised to identify patients with a neoplastic lesion suggesting potential application in the clinical setting. In this article, we present the scientific evidence to support field cancerisation in colorectal cancer and discuss important limitations that require further investigation. Characterisation of the field defect is necessary to enable early diagnosis of colorectal cancer and identify molecular targets for chemoprevention. Field cancerisation offers a promising prospect for experimental cancer research and has potential to improve patient outcomes in the clinical setting.
    • Splice variants of the extracellular region of RON receptor tyrosine kinase in lung cancer cell lines identified by PCR and sequencing

      Krishnaswamy, S; Mohammed, A.K; Tripathi, G; Alokail, M.S; Al-Daghri, N.M; University of Westminster (BMC, 09/11/2017)
      Altered expression of receptor tyrosine kinases (RTKs) is a major driver of growth and metastasis of cancers. Recepteur d’origine nantais (RON) receptor is a single-pass transmembrane RTK aberrantly expressed in a number of cancers. Efforts to block deregulated RON signaling in tumors using small molecule kinase inhibitors or antibodies are complicated by the presence of unknown number/types of isoforms of RON, which, despite having similar sequences, are localized differently and mediate varied functions. The objective of this study was to identify splice variants of RON transcripts between exons 1 and 10 that code for the extracellular region. Direct cDNA sequencing was performed for the transcript between exons 1–10 of RON by Sanger sequencing in various lung cancer cell lines. PCR amplification and bi-directional sequencing of cDNA for section between exons 1 and 10 from lung cancer cell lines revealed the presence of several splice variants of RON transcripts; the variants were formed by skipping of exons 2, 2–3, 5–6, 6 and 8–9. Each of these transcript variants were found in one or more cell lines. While the variants formed by skipping of exons 2, 2–3 and 5–6 resulted in loss of 63, 106 and 109 amino acids, respectively, and didn’t cause reading-frameshift, the transcripts formed by skipping of exons 6 and 8–9 caused reading-frameshift. Splice variant lacking exons 8–9 was found in 13 out of 23 cell lines tested. Lung cancer cell lines contain several splice variants of RON which involve skipping of exons coding for extracellular region. Some of the splicing changes result in reading-frameshift and the N-terminally truncated isoforms are expected to be secreted out. The ubiquitous nature of alternative splicing events in RON suggests the need for isoform specific approaches to functional analysis and therapeutic targeting of RON.