The effect of Cyclopia maculata on AMPK expression in Wistar rats

>Magister Scientiae - MScBeing overweight or obese are major factors contributing to the increased morbidity and mortality due to non-communicable diseases such as type 2 diabetes, cardiovascular disease and cancer. The treatment of obesity with pharmaceutical drugs is plagued by side effects. Plants and their phytochemicals possess a number of beneficial health effects including anti-oxidant,anti-mutagenic, anti-inflammatory, anti-obesity and anti-cancer effects, mediated by activation of the adenosine monophosphate protein kinase (AMPK).AMPK controls many metabolic processes including glucose uptake and utilisation, and adipogenesis, and is often referred to as the master regulator establishing cellular homeostasis.Cyclopia maculata, commonly known as honeybush, is an indigenous South Africa plant possessing anti-oxidant, anti-inflammatory and anti-cancer properties. Recently, others in our laboratory have shown that a hot water extract of fermented C. maculata inhibits adipocyte differentiation in 3T3-L1 pre-adipocytes, with some evidence of weight regulatory properties in a Wistar rat model of diet-induced obesity. In the rat study, 21 day old weanlings were fed a high fat, high sugar cafeteria diet for 3 months with (n=10) or without (n=10) C. maculata supplementation. This group of rats was referred to as the lean group (n=20). Another group of rats were fed a cafeteria diet for 4 months to induce obesity (obese group, n=20) and thereafter treated as described for the lean rats. The aim of this MSc study was to determine whether C. maculata induces AMPK activation.Proteins were extracted from the liver and muscle tissue of lean and obese Wistar rats using an optimized extraction method with a commercial lysis buffer and the TissueLyser.Treatment with the C. maculata extract had no effect on the protein yield in lean and obese rats. Interestingly, the protein yield in the liver of obese rats was significantly higher than that observed in lean rats. Although C. maculata treatment slightly increased AMPK activation (calculated as the ratio of phosphorylated AMPK to total AMPK) in the liver of lean and obese rats, the difference was not statistical significant. Conversely, C.maculata treatment decreased AMPK activity in muscle of lean and obese rats, with statistical significance observed in the lean group only (2.3-fold, p<0.05). Differences in AMPK activation between the groups were also noted, a 1.3-fold decreased activity observed in obese groups compared to their lean counterparts, although this was not statistically significant. Expression of PPARα, a downstream protein target affected by AMPK activation was reduced in the liver of lean and obese rats after C. maculata treatment. Moreover, PPARα expression was significantly higher in obese compared to lean rats (2.7-fold, p<0.001). PPARα is a transcription factor mediating fat metabolism (β-oxidation) and its expression is induced by circulating free fatty acids, which are increased in obese compared to lean rats. The expression of PPARα in muscle was too low for Western blot analysis and quantification.Cyclopia maculata treatment did not affect hepatic expression of UCP2, another protein important in establishing energy homeostasis. The expression of UCP2 was 2.9-fold higher in the liver of obese rats compared to their lean counterparts, although the difference was not statistically significant. The opposite results were observed in the muscle where C. maculata treatment decreased UCP2 expression in lean rats (2.8-fold,p<0.0001), and UCP2 expression was decreased 1.4-fold in obese rats compared to lean rats, although the difference was not statistically significant.ELISA results for AMPK activation revealed that C. maculata treatment increased AMPK activity, although not statistically significant. Histological analysis of retroperitoneal fat showed that C. maculata did not affect adipocyte size and number, although a slight decrease in adipocyte size was observed after treatment .This study has demonstrated that treatment of the cafeteria diet fed Wistar rats with 300 mg/kg of a hot water extract of fermented C. maculata does activate AMPK. This study revealed important differences between lean and obese rats. In particular, increased hepatic protein content, PPARα and UCP2 expression was observed in obese rats compared to the lean group. This suggests an adaptive response to the increased circulating free fatty acids during obesity and an increase in β-oxidation in these animals

Impact of HIV and antiretroviral drug exposure on lung growth and function over 2 years in an African Birth Cohort.

OBJECTIVE: To assess the impact of HIV and antiretroviral exposure without infection on lung growth and function over the first 2 years of life. DESIGN: Prospective observational study of an African birth cohort, Drakenstein Child Health Study. METHOD: Infants enrolled antenatally had lung function measured at 6 weeks, 1 and 2 years. HIV-infected women received antiretroviral therapy (ART) as per local guidelines. The association between HIV and antiretroviral exposure with lung function was assessed using mixed effects modelling. RESULTS: Of 1143 infants born, two HIV-infected infants were excluded from analysis; 909 (80%) infants had lung function collected at 6 weeks [190 (21%) were HIV-exposed uninfected (HEU)]; 782 (69%) at 1 year and 741 (65%) at 2 years. At 6 weeks HEU infants had larger tidal volume compared with HIV-unexposed infants (1.13?ml, confidence interval: 0.02-2.23, P?=?0.045). High maternal viral load was associated with a 17% lower expiratory flow over 2 years (0.17, confidence interval 0.00-0.34, P?=?0.046). First-line ART initiated during pregnancy was associated with lower infant tidal volume at 6 weeks compared with those who initiated ART before pregnancy (-2.7?ml, -5.31 to -0.10, P?=?0.042), and low maternal CD4 cell counts associated with lower infant tidal over 2 years (-11.1?ml, -18.58-3.58, P?=?0.004). CONCLUSION: HIV exposure is associated with altered lung function in early life, with a vulnerable HEU subgroup based on maternal disease severity, immunological compromise and ART exposure. These data highlight the importance of ongoing surveillance of respiratory health in HEU children

Optimized algorithm for speed-of-sound-based infant sulfur hexafluoride multiple-breath washout measurements.

INTRODUCTION Major methodological issues with the existing algorithm (WBreath) used for the analysis of speed-of-sound-based infant sulfur hexafluoride (SF6) multiple-breath washout (MBW) measurements lead to implausible results and complicate the comparison between different age groups and centers. METHODS We developed OASIS-a novel algorithm to analyze speed-of-sound-based infant SF6 MBW measurements. This algorithm uses known context of the measurements to replace the dependence of WBreath on model input parameters. We validated the functional residual capacity (FRC) measurement accuracy of this new algorithm in vitro, and investigated its use in existing infant MBW data sets from different infant cohorts from Switzerland and South Africa. RESULTS In vitro, OASIS managed to outperform WBreath at FRC measurement accuracy, lowering mean (SD) absolute error from 5.1 (3.2) % to 2.1 (1.6) % across volumes relevant for the infant age range, in variable temperature, respiratory rate, tidal volume and ventilation inhomogeneity conditions. We showed that changes in the input parameters to WBreath had a major impact on MBW results, a methodological drawback which does not exist in the new algorithm. OASIS produced more plausible results than WBreath in longitudinal tracking of lung clearance index (LCI), provided improved measurement stability in LCI over time, and improved comparability between centers. DISCUSSION This new algorithm represents a meaningful advance in obtaining results from a legacy system of lung function measurement by allowing a single method to analyze measurements from different age groups and centers

Normal values of respiratory oscillometry in South African children and adolescents

Introduction Noninvasive measurement of respiratory impedance by oscillometry can be used in young children aged from 3 years and those unable to perform forced respiratory manoeuvres. It can discriminate between healthy children and those with respiratory disease. However, its clinical application is limited by the lack of reference data for African paediatric populations. The aim of the present study was to develop reference equations for oscillometry outcomes in South African children and adolescents. Methods Healthy subjects, enrolled in the Drakenstein Child Health Study, HIV-uninfected adolescents in the Cape Town Adolescent Antiretroviral Cohort and healthy children attending surgical outpatient clinics at Red Cross War Memorial Children's Hospital were measured with conventional spectral (6–32 Hz) and intra-breath (10 Hz) oscillometry. Stepwise linear regression was used to assess the relationship between respiratory variables and anthropometric predictors (height, sex, ancestry) to generate reference equations. Results A total of 692 subjects, 48.4% female, median age of 5.2 years (range: 3–17 years) were included. The median (interquartile range (IQR)) for weight for age z-score and height for age z-score was −0.42 (−1.11–0.35) and −0.65 (−1.43–0.35), respectively. Stepwise regression demonstrated that all the variables were significantly dependent on height only. Comparison to previous reference data indicated slightly higher resistance and lower compliance values in the smallest children. Conclusion We established the first respiratory oscillometry reference equations for African children and adolescents, which will facilitate use in early identification and management of respiratory disease. Our results suggest differences in oscillometry measures by ancestry but also highlight the lack of standardisation in methodology