The effect of Cyclopia maculata on AMPK expression in Wistar rats
Jacobs, Carvern Denver
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Being 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.