Supplementation of phytochemicals in human diets has been found to provide numerous health benefits (Pan et al., 2009
). Açai pulp is rich in flavonoids, many of which have been shown to have potential health benefits in various test models (Schauss et al., 2006b
). Here we have evaluated the anti-aging effect of açai pulp supplementation in D. melanogaster
and investigated potential molecular mechanisms. By testing concentrations of açai up to 2% in the food, we have not observed any prolongevity or lifespan shortening effect of açai on flies fed a commonly used standard fly diet. Using an improved protocol to prepare high fat diets, we have found that a high fat diet containing palmitic acid, a saturated fatty acid, significantly decreases lifespan compared to a standard diet fed to controls. Açai supplementation significantly increases mean and, to a less extent, maximum lifespan of flies fed a high fat diet. This prolongevity effect appears to be associated with decreased expression of Pepck and an increase in stress response. In addition, we found that açai pulp improved survival of sod1
RNAi flies, further suggesting that açai pulp can alleviate oxidative damage in vivo
at the organismal level. Interestingly the phenotype of açai supplementation resembles that of supplementation of resveratrol, a polyphenolic compound with anti-aging effects, in mice (Howitz et al., 2003
). Resveratrol has been shown to prolong the lifespan of mice fed a high fat diet but not those fed a standard rodent diet (Baur et al., 2006
; Pearson et al., 2008
). However, resveratrol appears to improve healthspan, although not the lifespan, of mice fed the standard diet as indicated by its beneficial effects on cognitive function (Pearson et al., 2008
). It remains to be determined whether açai supplementation has any effect on healthspan of flies fed a standard fly diet.
Restriction of calorie intake without causing malnutrition is an effective non-genetic approach to extend lifespan in almost every animal model (Barzilai and Bartke, 2009
). A possible mechanism of the prolongevity effect of açai for flies fed the high fat diet is that it may suppress food intake and indirectly increase lifespan through calorie restriction (CR) pathways. This is not likely the scenario for the action of açai. Firstly, açai supplementation up to 2% does not increase lifespan of flies fed the normal diet. This suggests that any potential effect of açai at the levels tested in this study on food intake is not sufficient to extend lifespan. Secondly, açai supplementation does not decrease the lifespan of flies fed the normal diet, either, indicating that açai at the concentrations used in this study is not detrimental to longevity. Thirdly, a comprehensive analysis of the nutrient composition indicates that freeze-dried açai pulp contains approximately 1.3 g sugar, 8.1 g protein and 32.5 g fat per 100 gram dry mass (Schauss et al., 2006a
). The high fat diet used in this study contains 10g sugar, 10g protein and 2 g fat per 100 mL food. Supplementation of açai to 2% in this diet will increase sugar, protein and fat in the food by 0.03 g, 0.16 g and 0.64 g per 100 mL food, which only slightly increases the calorie content of the diet (<7% increase). Most critically, food intake with 24 hrs was found not significantly different between female flies on the high fat control diet and the high fat diet supplemented with 2% açai. Taken together, açai supplementation has a minimal impact on calorie content of the fly diets and does not appear to promote longevity through suppressing food intake of the flies under the culture conditions used in this study.
Pepck is a key enzyme controlling gluconeogenesis and glyceroneogenesis, and known to be modulated by a number of transcription factors, including dFoxo (Chakravarty et al., 2005
). dFoxo is a forkhead transcription factor and is regulated at the protein phosphorylation level by the insulin/insulinlike signalling pathway, a major pathway involved in regulating metabolism and aging (Kenyon, 2005
). We have observed that açai supplementation significantly suppresses Pepck
expression, but does not alter the transcript level of dFoxo
. dFoxo is thought to be an transcriptional activator of Pepck
(Chakravarty et al., 2005
). Taken together, this suggests that açai modulates Pepck expression without changing the transcript level of dFoxo. However, we cannot rule out the possibility that açai modulates phosphorylation and nuclear entry of dFOXO protein, which in turn regulates Pepck expression in this study. It also remains to be determined whether the change of Pepck expression influences lifespan.
Our molecular analysis has revealed that açai supplementation restores the transcript level of l(2)efl
in flies fed a high fat diet to that of flies fed a normal diet. l(2)efl
encodes a small heat-shock-related protein and is known to be activated by the JNK signaling pathway, a major pathway that responds to various stresses, including oxidative stress (Wang et al., 2005
). Oxidative damage is thought to be one of the causative factors in aging (Colavitti and Finkel, 2005
; Perez et al., 2009
). Activation of the JNK signaling pathway has been shown to extend lifespan in D. melanogaster
, which is partially mediated by l(2)efl
, a JNK target (Wang et al., 2005
). Overexpression of l(2)efl
in whole body or in neurons only can extend lifespan in D. melanogaster
(Wang et al., 2005
). Although most of the genes tested in this study, including JNK, do not show significant alterations at transcript levels, we can not rule out the possibility that their protein activities are modulated by açai supplementation. Analyses of six additional putative downstream genes of the JNK signaling pathway showed that supplementation of açai elevated expression of two of them, GstD1
, which are involved in detoxification, compared to the high fat control. In addition, we found that açai pulp increased the lifespan of oxidative stressed flies induced by sod1
RNAi. Although l(2)efl
are regulated by other signaling pathways, our findings suggest açai pulp modulates lifespan at least partially through activation of the JNK signaling pathway.
Our findings suggest that açai pulp may be effective in prevention and control of type 2 diabetes. Chronic high fat diets have been shown to lead to insulin resistance and eventually type 2 diabetes. The effectiveness of some diabetic drugs, such as fenofibrate, appears to be mediated by their abilities to suppress Pepck
expression and, in turn, gluconeogenesis and glyceroneogenesis (Chakravarty et al., 2005
; Srivastava, 2009
). This suppression can lower the glucose levels and improve lipid profiles in type 2 diabetic patients and animal models, which results in effective control of diabetic symptoms (Quinn and Yeagley, 2005
). We have found that açai pulp reduces the transcript level of Pepck
by more than four fold compared to the non-supplemented diets. This suggests that açai pulp can antagonize the detrimental effects of high fat diets as a functional food. Further animal model and clinical studies need to be conducted to investigate the effects of açai pulp consumption in prevention of diseases, including diabetes.