Knockdown of Indy/CeNac2 extends Caenorhabditis elegans life span by inducing AMPK/aak-2.

Reducing the expression of the Indy (Acronym for 'I'm Not Dead, Yet') gene in lower organisms promotes longevity and leads to a phenotype that resembles various aspects of caloric restriction. In C. elegans, the available data on life span extension is controversial. Therefore, the aim of this study was to determine the role of the C. elegans INDY homolog CeNAC2 in life span regulation and to delineate possible molecular mechanisms. siRNA against Indy/CeNAC2 was used to reduce expression of Indy/CeNAC2. Mean life span was assessed in four independent experiments, as well as whole body fat content and AMPK activation. Moreover, the effect of Indy/CeNAC2 knockdown in C. elegans with inactivating variants of AMPK (TG38) was studied. Knockdown of Indy/CeNAC2 increased life span by 22±3 % compared to control siRNA treated C. elegans, together with a decrease in whole body fat content by ~50%. Indy/CeNAC2 reduction also increased the activation of the intracellular energy sensor AMPK/aak2. In worms without functional AMPK/aak2, life span was not extended when Indy/CeNAC2 was reduced. Inhibition of glycolysis with deoxyglucose, an intervention known to increase AMPK/aak2 activity and life span, did not promote longevity when Indy/CeNAC2 was knocked down. Together, these data indicate that reducing the expression of Indy/CeNAC2 increases life span in C. elegans, an effect mediated at least in part by AMPK/aak2.


INTRODUCTION
Na-sulfate cotransporters in vertebrates, invertebrates, plants, and bacteria [10,11]. mINDY mediates the cotransport of citrate, succinate, and several other dicarboxylates across the plasma membrane together with sodium in an electrogenic manner [12]. The amino acid sequence of the N-terminal sodium and the carboxy-binding motif is highly conserved between many species, from bacteria to C. elegans to human [13]. Therefore, C. elegans might serve as a model entity for higher organisms.
The C.elegans Indy homolog CeNAC2 is expressed mostly in tissues that function as sites of nutrient absorption and fat storage [4,12,14]. Indy/CeNAC2 is located on the plasma membrane and transports TCA cycle intermediates, which are used to generate biochemical energy in the form of ATP. A tight regulation of net energy flux is a fundamental requirement of living organisms. Alteration of these processes can lead to profound changes in the ability of cells to cope with sudden changes in energy demand and can result in premature death. Therefore, cells possess several mechanisms to adjust and tune in energy demand and synthesis. One kinase controlling these mechanisms is AMP activated protein kinase (AMPK) [15,16]. AMPK is activated in states of low energy supply through AMP, and it is inhibited in states of high energy abundance by ATP. Upon activation, AMPK increases mitochondrial biogenesis and shifts energy generation from glycolysis to the oxidation of fat, yielding higher ATP levels. By these and other mechanisms, the AMPK homolog aak2 mediates the life extending effect of glucose restriction in C. elegans [15,17,20].
Here, we aimed at clarifying whether or not knocking down the C. elegans Indy homolog CeNAC2 increases life span similar to the studies in D. melanogaster. We hypothesized that longevity in Indy/CeNAC2 reduced C. elegans is mediated via the activation of AMPK/aak2.

Effect of Indy/CeNAC2 knockdown on life span
Knockdown of Indy/CeNAC2 by feeding wildtype N2 worms with bacteria expressing Indy/CeNAC2-specific siRNA caused a significant increase in average life span of the organism (Fig. 1A). Individual data from 4 independent experiments are given in   (Fig. 1B-D).

Knockdown of CeNAC2 leads to the activation of AMPK
Given the life span extending effect of Indy/CeNAC2 knockdown together with a reduction in body fat content, we were interested in determining a unifying molecular mechanism. In mice, deletion of mIndy reduced the hepatocellular ATP/ADP ratio and activated hepatic AMPK [6,19]. The C. elegans AMPK homolog aak2, has been shown to mediate the life span extending effect of various short-term stressors [17] including glucose restriction [20]. Phosphorylation of AMPK/aak2 at threonine 172 is widely used as a surrogate marker of activity of this kinase. Therefore, the phosphorylation of aak2 in worms fed on bacteria harboring Indy/CeNAC2 siRNA was analyzed. Western blot analysis of cell extracts was carried out using an antibody against phospho-Thr 172 of the human AMPK. AMPK/aak2 was markedly phosphorylated in worms with reduced Indy/CeNAC2. A lower state of phosphorylation of AMPK/aak2 was observed for wildtype N2 under control conditions, fed with bacteria harboring the empty vector L4400. Blotted extracts of worms with a deletion in the aak2 gene (TG38 aak-2(gt33)), including the phosphorylation site, showed no signal for phosphorylated AMPK/aak2 (Fig. 1E).

Role of AMPK/aak2 in the life span extending effect of Indy/CeNAC2
In order to determine whether or not the life span extending effect of the reduction of Indy/CeNAC2 depends on AMPK/aak2, life span studies were repeated in C. elegans with an inactivating mutation of AMPK/aak2, TG38 aak-2(gt33). In these TG38 aak-2(gt33) worms, RNAi-mediated knock down of Indy/CeNAC2 did not increase average life span, while we continued to observe the life span extending effect in the N2 wild type ( Fig.2A

DISCUSSION
Our studies show that knockdown of Indy/CeNAC2 in C. elegans leads to a significant increase in life span. AMPK/aak2 was activated in CeNAC2 reduced C.  A previous study did not observe a life span extending effect of Indy/CeNAC2 knockdown in C. elegans nor a life span increase with Indy knockdown in D. melanogaster [9]. Subsequent studies in D. melanogaster have resolved this discrepancy in flies by demonstrating that knockdown of Indy in flies is mediated by the induction of a calorie restriction-like state [4], as it is in mice [6]. A possible explanation might be that the two studies used different diets, with the study that did not observe a life span extension used a diet in flies that resembled more a low calorie diet [4,9,22]. In this scenario, it is possible that the knockdown of Indy does not extend life span any further.
In the study presented here, E.coli GC363 harboring the empty vector L4400 or the vector with the CeNAC2specific siRNA was used. Using this regular, commonly used food [23], we observed a life span extending effect with the knock down of Indy/CeNAC2 in C. elegans for mean, median and maximum life span (see supplemental data Table S3 and S5). Together with life span extension, Indy/CeNAC2 Knockdown also resulted in reduced whole body fat stores in our worms. This finding is in line with observations in D. melanogaster [4], C. elegans [8] and mice [6]. The reduction in body fat might be mediated via multiple mechanisms. First, the functional role of Indy/CeNAC2 is to import citrate as well as several dicarboxylates into the cytosol as substrates for several metabolic processes [8]. For example, citrate is the main carbon source of fatty acid and cholesterol synthesis, and it acts as an allosteric activator of acetyl-CoA carboxylase (ACC) [11]. Moreover, cytosolic citrate furnishes NADPH generation via malic enzyme for lipogenesis [24]. Overexpression of mammalian Indy homolog in a HepG2 cell line resulted in an increase in intracellular fatty acid and sterol synthesis in our previous studies [25]. In line with this notion, enhancing the activity of citrate transport by mINDY in HepG2 cells has been shown to enhance citrate induced lipid synthesis [25,26], while knocking down mINDY in hepatocytes leads to a reduction in hepatocyte lipid content [6,27]. It is possible that reducing the import of citrate by knocking down Indy/CeNAC2 reduces lipid synthesis directly by reducing citrate as a C-donor for fatty acid synthesis as well as an allosteric activator for lipid synthesis [27].
Moreover, we observed that reducing Indy/CeNAC2 expression is associated with the activation of the intracellular energy sensor AMPK/aak2. When reducing Indy/CeNAC2 expression in AMPK/aak2 mutated C. elegans TG38 aak-2(gt33), which do not possess the ability to phosphorylate, and thus, activate AMPK, the life span extending effect of the Indy/CeNAC2 KD was not observed any longer.
AMPK has previously been shown to mediate longevity in C. elegans when glucose is restricted as a nutritional substrate [20]. In this case, AMPK is activated via AMP. Moreover, AMPK has also been put into context to mediate the longevity effect of SIRT1 and metformin in C. elegans and mice [15,16,28]. Moreover, metformin might activate AMPK via inhibition of complex 1 of the respiratory chain, reducing ATP generation and increasing the AMP/ATP ratio. Other mechanisms contribute [29]. The inability to increase lipid oxidation in aging rats has been attributed to reduced activation of AMPK [30]. Finally, in mice with deleted mINDY, the ATP/AMP ratio is shifted towards AMP, and ATP levels are largely reduced [6,31]. Consecutively, AMPK is activated when INDY is knocked out in mice. Collectively, these data provide a rationale for the molecular mechanisms mediating longevity when Indy/CeNAC is reduced.
In our studies, reducing glucose availability through DOG did not enhance life span any further in Indy/CeNAC2 knock down, as shown for mean, median and maximum life span (see supplemental data Table S4 and S6). These data further strengthen the role of AMPK in mediating the life span extending effect of Indy/CeNAC2 reduction, since inhibition of glycolysis using DOG in C. elegans has been shown to lead to an extended life span by activating AMPK [20]. Findings from Neretti and colleagues suggested that when INDY is reduced, a decreased flux of electrons through the electron transport chain associated with an upregulation of mitochondrial biogenesis combine to result in an overall decrease of reactive oxygen species and maintenance of normal cellular ATP levels [1]. Clearly, a thorough analysis of the metabolic pathways that are altered by the deletion and/or reduction of Indy/CeNAC2 are needed to better understand how AMPK/aak2 might be activated.
In summary, knockdown of Indy/CeNAC2 in C. elegans extends life span along with the activation of the intracellular energy sensor AMPK. Our data support the hypothesis that AMPK is indispensable for Indy/CeNAC2 to mediate longevity and provide a rationale for the molecular mechanisms mediating longevity when Indy/CeNAC2 is reduced. Together, www.impactaging.com these data suggest that reducing the expression or activity of the mammalian homolog of the tri-and dicarboxylic acid transporter Indy/CeNAC2 might be an attractive target to promote healthy aging by activating AMPK and, possibly, to extend life span.

METHODS
Strains and culture conditions. C. elegans strains Wildtype N2, TG38 aak-2(gt33) and E.coli OP50 and GC363 were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN). The gt33 allele has a 606-bp deletion. Wild-type N2 and aak-2 mutant worms were maintained at 22°C on Nematode Growth Medium (NGM) plates seeded with E. coli OP50 using standard methods [32] except for experiments where other is specified.
Bacterially-mediated RNAi. Genetically modified bacteria strain (E. coli HT115 ) for CeINDY was retrieved from Source BioScience. Carrying the L4400 vector, which contains convergent T7 polymerase promoters separated by a multicloning site. E. coli GC363 carry the empty L4400 vector and are considered as the control. Control and siRNA expressing E.coli strains were grown over night on LB Agar at 37°C containing 100 µg/ml ampicillin and 12.5 µg tetracyclin. A single colony was picket and transferred into LB-Media and cultivated over night for a starter culture. Preceding every experiment for bacterial-mediated RNAi, a fresh culture was grown for 6h in LB-Media from an aliquot of the starter culture. 4-7 days ahead of every experiment the according NGM plates were prepared containing 100 µg/ml ampicillin (Roth) and 1mM Isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma) as well as 0. Statistical analysis. Statistical analyses for Indy/CeNAC2 knock down and fat content were performed by Wilcoxon test. For comparing significant distributions between different groups in the life span assays statistical calculations were carried out using the log-rank rest (Kaplan Meier) as well as the χ 2 -Test. Given data shows mean values ±SEM if not described otherwise. All calculations were performed using Microsoft Office Excel 2010 and SPSS version 20.0. Experiments were performed in triplicate except where stated otherwise. Error measures were calculated form the variability of the independently prepared biological samples of the same treatment group where applicable. Differences were considered statistically significant at p < 0.05, therey indicating a probability of error lower than 5%.