Fat accumulation in Caenorhabditis elegans triggered by the electrophilic lipid peroxidation product 4-Hydroxynonenal (4-HNE)

Results

Expression of the gst‑10 gene product, which is capable of conjugating 4‑HNE, is inversely related to the level of whole-body 4‑HNE-protein adducts

As we have previously shown [20,21], the capacity of C. elegans to conjugate 4‑HNE with glutathione was reduced by RNAi-mediated knockdown of CeGSTP2-2, the product of the gst‑10 gene. The loss of 4‑HNE-conjugating activity was only partial because of the existence of multiple GSTs capable of conjugating 4‑HNE [20]. Nevertheless, silencing of gst‑10 resulted in a greater than 50% increase of the level of whole-body 4‑HNE-protein adducts (Figure 1). Conversely, overexpression of gst‑10 results in an enhanced capacity to conjugate 4‑HNE and in a decrease in the content of 4‑HNE-protein adducts [22]. Because the biological effects of 4‑HNE are thought to be mediated mostly by formation of covalent adducts with proteins, a process that frequently alters protein function (reviewed in refs. [4, 6-9]), the observed changes in adduct amounts could be functionally significant.

Figure 1. Increased level of 4‑HNE-protein adducts in C. elegans with silenced gst‑10 gene. RNAi silencing of gst‑10 was carried out as previously described [21] in C. elegans strain Bristol-N2; worms fed bacteria transformed with an insert-free L4440 feeding vector served as the control. The level of 4‑HNE-protein adducts was determined in 5-day-old worms by competitive ELISA [93]. The values shown are means ± S.D. of 4 biological replicates (independently grown batches of worms) per group.

Fat accumulation in C. elegans is inversely proportional to the expression level of gst-10

Worms subjected to RNAi against gst‑10 accumulate significant amounts of fat (Figures 2A and 2B). The use of two independent lipid stains, Nile red [23,24] and Sudan black [25], minimizes the likelihood of artifacts. In contrast to animals subjected to RNAi, several C. elegans lines overexpressing the gst‑10 gene product [22] had a lean phenotype (Figure 2C). This lean phenotype was confirmed using a second, independently derived control line carrying an insert-free vector (data not shown), thus establishing that the low fat content of gst-10-overecxpressing worms was not due to an aberrant control line. The data demonstrate an inverse proportionality between gst‑10-linked 4‑HNE-conjugating activity and fat accumulation in C. elegans.

Figure 2. Lipid content of C. elegans with altered expression of gst‑10. (A) and (B): worms fed bacteria carrying the insert-free L4440 feeding vector (control) and worms subjected to RNAi against gst‑10. Lipid content was measured by Nile red fluorescence (A) and by Sudan black staining (B). (C): lipid content (by Sudan black) in worms transformed with the insert-free pPD49.26 vector (control) and in four transgenic C. elegans lines that overexpress gst‑10. Bars represent means ± S.D. (n = 50 worms per group) of pixel intensities integrated on worm micrographs.

Figure 3. Effects of RNAi against aldehyde dehydro-genases alh-1 and alh-10. (A) Effect of RNAi against alh-1 and alh-10 on NAD⁺-dependent 4‑HNE oxidation capacity of worm homogenates. (B) and (C): Fat content in C. elegans subjected to RNAi against alh-1 and alh-10, determined by Nile red (A) and Sudan black (B) staining followed by pixel integration. Means ± S.D. (n = 51 worms per group) are shown. In all experiments, statistical significance versus insert-free L4440 feeding vector control was assessed by the t‑test with Bonferroni correction.

Figure 4. Increased level of 4‑HNE-protein adducts in C. elegans treated with 4‑HNE. Synchronized L4 worms were kept in liquid culture for 3 days in medium containing 1 mM 4‑HNE. The medium was replaced every 12 hr with fresh medium containing 4‑HNE. The level of 4‑HNE-protein adducts was determined in 5-day-old worms by competitive ELISA [93]. The values shown are means ± S.D. of 4 biological replicates (independently grown batches of worms) per group.

Discussion

Experimental manipulation of enzymes that catalyze elimination of 4‑HNE from tissues modulates the steady-state level of 4‑HNE and affects the amount of stored fat in C. elegans. Specifically, inhibition of either conjugation or oxidation of 4‑HNE leads to increased fat accumulation, whereas overexpression of a 4‑HNE-conjugating enzyme results in a lean phenotype (Figure 2 and Figure 3). These observations are consistent with the obese phenotype we have previously reported for mGsta4 null mice which have an impaired ability to conjugate 4‑HNE [19], indicating that the link between abundance of 4‑HNE and fat storage is conserved between evolutionarily distant species.

We propose that 4‑HNE, rather than another substrate or function of the enzymes that are being experimentally manipulated, is a causative factor in the enhanced storage of lipid. We favor this model for the following reasons:(i) Except for 4‑HNE, there is no known common substrate of the two rather dissimilar C. elegans enzymes used in the present work, i.e., the products of gst‑10 and alh‑1 genes. Moreover, 4‑HNE is the only known common substrate of the above-mentioned two enzymes and the murine mGSTA4-4 [30].

(ii) Fat deposition was reported for yeast directly treated with 4‑HNE [31], an experimental design that points to 4‑HNE as the causative agent.

(iii) mGsta4 null mice in the 129/sv genetic background have an elevated tissue levels of 4‑HNE and are obese, while mGsta4 null mice in C57BL genetic background have near-normal levels of 4‑HNE and are not obese [19]. The proportionality of 4‑HNE and fat deposition in otherwise closely related systems suggests a causal role of 4‑HNE in lipid accumulation in the murine model.

(iv) Overexpression of murine aldose reductase Akr1b7 [an enzyme catalyzing reductive metabolism of 4-HNE, ref. 32] inhibits adipogenesis, and disruption of the same enzyme accelerates adipogenesis [33]. Concordant results obtained with mGSTA4-4 and Akr1b7 indicate that a common substrate, such as 4‑HNE, is responsible for the phenotype.

While alternative explanations could be invoked for each of the findings listed above, a strong case for a causal involvement of 4‑HNE can be made if the arguments are considered in aggregate. It is unlikely that several enzymes that act on distinct functional groups of 4‑HNE would all share a hypothetical additional substrate. The reactions these enzymes catalyze include Michael addition of a nucleophile to a polarized double bond (GSTs), NAD⁺-dependent oxidation of an aldehyde to a carboxylic acid (aldehyde dehydrogenase), and NAD(P)H-dependent reduction of an aldehyde to an alcohol (aldose reductase). To be able to undergo all three reactions, a substrate must contain the requisite target functional groups; this requirement restricts the set of possible substrates to α,β-unsaturated carbonyl compounds such as 4‑HNE. Moreover, experimental interventions that lower the expression of 4‑HNE-conjugating GSTs in C. elegans (gst-10) and in mice (mGSTA4-4) resulted in a similar phenotype, i.e., increased fat storage. The two enzymes are not orthologs, and their ability to conjugate 4‑HNE probably evolved independently [22,34]. It is unlikely that both enzymes acquired by chance another shared activity or function that is unrelated to conjugation of 4‑HNE, indicating that the parallel effects of disruptingmGSTA4-4 and gst-10 are indeed mediated by 4‑HNE.

Figure 5. Effects of direct treatment with 4‑HNE on lipid accumulation in C. elegans. Synchronized L4 worms were kept in liquid culture for 3 days in medium containing the indicated concentration of 4‑HNE. The medium was replaced every 12 hr with fresh medium containing 4‑HNE. (A) fluorescence of worms treated with the indicated concentration of 4‑HNE but not stained (upper row of images), and animals exposed to 4‑HNE and stained with Nile red (lower row of images). (B) brightfield images of worms treated with the indicated concentration of 4‑HNE and directly photographed (upper row of images), or photographed after staining with Sudan black (lower row of images). (C): for each 4‑HNE concentration, lipid content was measured in three independently treated worm populations by quantitation of Nile red fluorescence (upper plot) or Sudan black staining (lower plot).

As outlined above, experimental modulation of 4‑HNE-metabolizing enzymes indicates that 4‑HNE can trigger fat accumulation. However, even though highly persuasive, the evidence is indirect. The existence of an alternative substrate of the three 4‑HNE-metabolizing enzymes is unlikely but cannot be ruled out with certainty. In addition, decreasing the expression of the three enzymes could lead to lipid accumulation via three independent mechanisms, none of them involving 4‑HNE - a hypothesis that is implausible but not impossible. Therefore, we wished to test a possible causative role of 4‑HNE without relying on enzymes. For this purpose, we took advantage of the unique experimental opportunities afforded by C. elegans, a multicellular animal that can be handled by methods developed for microorganisms or cultured cells [35]. This includes the option to grow C. elegans in liquid medium that can be supplemented with compounds to be tested. Exposure of C. elegans to synthetic 4‑HNE led to dose-dependent lipid deposition (Figure 5) similar to that triggered by silencing of either gst-10 or alh-1. While this finding does not formally rule out the possibility that hypothetical alternative substrates of gst-10 and alh-1 are also involved, it proves that 4‑HNE is sufficient to elicit the fat accumulation phenotype.

Figure 6. Effect of increased 4‑HNE levels on malonyl-CoA concentration. C. elegans were either treated with 1 mM 4‑HNE according to the protocol described in the legend of Figure 5(A) or subjected to RNAi to gst‑10(B)). The results were normalized to the respective control and are shown as mean ± S.D. of 3 independently grown worm populations. In (A), the control (C. elegans Bristol-N2 in liquid culture in the presence of E. coli OP50 but without 4‑HNE) contained 16.5 ± 1.2 ng malonyl-CoA/mg dry weight; in (B), the level of malonyl-CoA in the control (C. elegans Bristol-N2 grown on plates seeded with E. coli HT115(DE3) transformed with insert-free L4440 feeding vector) was 12.2 ± 0.8 ng/mg dry weight.

The increase in stored fat was similar for worms directly exposed to 4‑HNE (Figure 5) and for those with silenced 4‑HNE-metabolizing enzymes (Figure 2 and Figure 3), even though the former treatment was markedly more effective in raising the level of 4‑HNE-protein adducts (Figure 4 versus Figure 1). These findings could indicate that silencing gst‑10 or alh-1 raises the concentration of 4‑HNE in selected tissues, including those involved in regulating fat storage. In contrast, exposure of animals to external 4‑HNE is likely to affect the entire body. Either treatment would reach the critical target tissues and trigger fat accretion, but exposure to 4‑HNE in the medium would generate more total 4‑HNE adducts. Further work is needed to identify the cells or tissues that are requires for increased fat storage in response to 4‑HNE, although head neurons are a possibility. Head neurons express the gst‑10 [22] as well as the alh-1 gene products (WormBase release WS194).

What is the molecular mechanism by which 4‑HNE increases the level of stored fat? A clue is offered by our finding that the concentration of malonyl-CoA was increased in C. elegans with disrupted enzymatic removal of 4‑HNE, as well as in animals directly exposed to 4‑HNE (Figure 6). Malonyl-CoA has a dual metabolic function: it is the substrate for fatty acid synthesis, and it prevents fatty acid β-oxidation [reviewed in ref. 36]. Therefore, elevated malonyl-CoA levels lead to an accumulation of fatty acids and, consequently, of triglycerides. In fact, experimental lowering of malonyl-CoA levels results in a lean phenotype in mammals [37-39] as well as in C. elegans [40]. Conversely, liver-specific disruption of AMPK, expected to increase malonyl-CoA levels, results in elevated hepatic lipogenesis [41,42]. These data indicate that the higher level of malonyl-CoA that we observed in worms with increased 4‑HNE could account for the concomitant fat accumulation.

Malonyl-CoA is formed from acetyl-CoA by the enzyme acetyl-CoA carboxylase (ACC). Although the steady-state tissue concentration of malonyl-CoA depends on the rate of its synthesis by ACC as well as its removal rate by fatty acid synthase or by malonyl-CoA decarboxylase, the activity of ACC is considered to be the major physiological determinant of malonyl-CoA levels at least in some tissues [43]. Therefore, the regulation of ACC activity is of central importance for fat homeostasis. Not surprisingly, ACC has been proposed as a target for pharmacological intervention [39,44].

Regulation of ACC is mostly post-translational. In mammals, a major regulatory mode is phosphorylation, particularly by AMPK [45,46]. Although C. elegans ACC lacks the canonical target sequence for AMPK phosphorylation [47], worm ACC is probably still regulated by AMPK, perhaps using an alternative phosphorylation site, because AMPK is required for metabolic modulation of fat stores [48]. Further work will be needed to elucidate the details of ACC phosphorylation in C. elegans.

In addition to phosphorylation, ACC is acutely regulated by allosteric effectors including citrate (reviewed in refs. [47, 49]). In the well-fed state, excess citrate is withdrawn from the TCA cycle and transported out of mitochondria. In the cytosol, citrate has a dual function in fat synthesis: (i) it allosterically activates ACC, and (ii) it is converted by citrate lyase to acetyl-CoA, the substrate of ACC (Figure 8). The resulting higher flux through ACC increases the supply of malonyl-CoA.

Figure 7. Effect of increased 4 HNE levels on citrate concentration. C. elegans were either treated with 1 mM 4- HNE according to the protocol described in the legend of Figure 5(A) or subjected to RNAi to gst 10 (B). The results are shown as means ± S.D. of 3 to 6 independently grown worm populations.

A partial inhibition of the tricarboxylic acid cycle enzyme aconitase lead to increased steady-state levels of citrate [50]. This is relevant to the present work because aconitase is susceptible to inhibition by 4‑HNE. It has been reported that adducts of 4‑HNE with aconitase form when isolated mitochondria are exposed to 4‑HNE [ref. 51 and references therein], and that treatment of cultured cells with 4‑HNE leads to a decrease in aconitase activity [52]. We found that mGsta4 knockout mice have lower aconitase activity and higher citrate levels than wild-type animals [19]. We now demonstrate that increasing the levels of 4‑HNE, either by direct treatment with the compound or by disruption of the metabolic disposal of 4‑HNE, results in an elevated citrate concentration in C. elegans (Figure 7). Thus, our results support a model in which 4‑HNE partially inhibits aconitase, causing an increase in the steady-state concentration of citrate. The latter metabolite augments the flux through ACC by providing both the substrate and the allosteric activator of the enzyme. This leads to more malonyl-CoA production, and thus to more fatty acid synthesis and less fatty acid β-oxidation. The accumulating fatty acids are converted to storage fat. The model is schematically depicted in Figure 8.

We propose that the chain of events leading from 4‑HNE to increased fat storage operates not only when 4‑HNE levels are experimentally perturbed, but that it is part of normal physiology. In the wild, access to food is typically intermittent. It is therefore highly adaptive to maximize stores of metabolic fuel when food is available. Such stores increase the probability of surviving "lean" periods. Feeding triggers the synthesis and deposition of fat. At least in mammals, increased fat storage correlates with a more pronounced pro-inflammatory and pro-oxidative state [53, 54]. Oxidative stress is expected to facilitate lipid peroxidation and raise 4‑HNE levels. In fact, it has been shown directly that feeding-induced obesity in mice causes an increase in tissue concentration of 4‑HNE [14]. In turn, elevated 4‑HNE realigns metabolism to favor fat deposition by increasing malonyl-CoA production, as described by us in the present report. We propose that these two partial processes - fat-induced 4‑HNE generation and 4‑HNE-triggered fat accumulation - form a positive feedback loop that perpetuates lipid storage as long as food is available. The proposed self-reinforcing loop is schematically depicted in Figure 8.

Figure 8. Proposed role of 4‑HNE in the generation and maintenance of fat stores.

Although self-sustaining while food is available, the postulated regulatory circuit involving 4‑HNE is not irreversible. When food becomes scarce (i.e., under caloric restriction conditions), metabolic and sensory inputs would act to shift metabolism to a mode that emphasizes somatic maintenance and utilization rather than storage of fat [55-57]. In this physiologic state, ROS generation is decreased, perhaps because of altered mitochondrial function [58]. Less ROS translates into less 4‑HNE production. At the same time, decreased insulin-like signaling would augment the Daf-16-dependent expression of the gst‑10 gene that encodes a 4‑HNE-conjugating enzyme [21,22]. The combination of limited supply and enhanced removal would lower the concentration of 4‑HNE, thus decreasing the production of malonyl-CoA by reversing the mechanisms summarized in Figure 8. This would, in turn, limit fatty acid synthesis and permit fatty acid β-oxidation, as required for the utilization of fat as a metabolic fuel.

The mechanism proposed in Figure 8 suggests that the metabolic "pro-adipose bias" observed in the well-fed state can be reversed not only by withdrawal of food, but also by withdrawal of 4‑HNE, even in the continuing presence of excess food. Pharmacological agents that act as 4‑HNE scavengers have been reported [59-64]. Clearly, the 4‑HNE-mediated mechanism we propose is not the only process that favors fat accumulation. Nevertheless, the potential importance of modulating 4‑HNE levels in preventing or treating obesity could be considerable.

Modulation of gst‑10 expression has opposite effects on fat accumulation and on life span of C. elegans. Worms with silenced gst‑10 (and elevated 4‑HNE) accumulate fat (Figure 2) and have a shortened life span [21], whereas overexpression of gst‑10 results in a lean phenotype (Figure 2) and it prolongs life [22]. In mammals, on an epidemiological level obesity correlates with an increased risk for metabolic syndrome and earlier mortality [reviewed in ref. 65]; it has been recently suggested that oxidative stress-triggered fat deposition not only correlates with, but causes aging [66]. However, the link between fat accumulation, disease, and longevity, while undeniable, is complex and defies simple generalizations. The tissue distribution of lipid is an important factor. Recent data indicate that the primary trigger of metabolic syndrome in mammals is not total but ectopic fat, and that fat in adipose tissue may play a lesser role or could actually be protective by preventing the accretion of ectopic fat [65,67-71]. Another factor that is relevant to longevity is the chemical nature of lipids, in particular the degree of unsaturation of fatty acids and the position of double bonds; highly peroxidizable polyunsaturated fatty acids may limit life span [72-77]. In C. elegans, fat accumulation has been found to be associated with either shortened or extended life span [48,78-82], probably reflecting differences in fat localization, structure, or timing of fat abundance in the course of development. Recent work has demonstrated that lipid stores provide a metabolic link in the signaling pathway from germline stem cells to longevity, as well as in the mechanism by which lower Daf-2 activity leads to an extension of life [83]. In the latter work, the decreased fat stores that were associated with a longer life span were caused by induction of a triglyceride lipase. We report the same inverse relationship between fat and longevity when lipid stores are modulated by altering the supply of fatty acids [this work and refs. 20-22]. Therefore, our data support the notion that accumulation of fat, whether through increased synthesis or lowered utilization, can shorten life span of C. elegans.

In summary, we have identified a biochemical mechanism, mediated through the lipid peroxidation product 4‑HNE, that promotes fat accumulation. The mechanism is part of a positive feedback loop that confers a pro-adipose bias on organismal lipid homeostasis. The proposed mechanism differs from the better-known endocrine regulation of fat-related physiology by its purely biochemical nature. Therefore, the mechanism may offer heretofore unexplored opportunities for intervention.

Acknowledgments

This work was supported in part by National Institutes of Health grants R01 AG028088 and AG018845 (to P.Z.). P.Z. is a recipient of a VA Research Career Scientist Award.

Conflicts of Interest

The authors declare no conflict of interest.

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