This AOP is licensed under a Creative Commons Attribution 4.0 International License.
Antagonist binding to PPARα leading to body-weight loss
- Kurt A. Gust
|Author status||OECD status||OECD project||SAAOP status|
|Open for citation & comment||TFHA/WNT Endorsed||2.3||Included in OECD Work Plan|
This AOP was last modified on August 16, 2019 10:18
|Decreased, PPARalpha transactivation of gene expression||April 30, 2019 12:05|
|Decreased, Ketogenesis (production of ketone bodies)||April 30, 2019 12:40|
|Not Increased, Circulating Ketone Bodies||April 30, 2019 12:45|
|Decreased, Body Weight||April 30, 2019 12:54|
|Increased, Catabolism of Muscle Protein||April 30, 2019 12:48|
|Binding of antagonist, PPAR alpha||September 16, 2017 10:14|
|stabilization, PPAR alpha co-repressor||April 30, 2019 11:42|
|Fatty Acid Beta Oxidation, Decreased||April 30, 2019 12:22|
|Binding of antagonist, PPAR alpha leads to stabilization, PPAR alpha co-repressor||June 11, 2018 22:49|
|stabilization, PPAR alpha co-repressor leads to Decreased, PPARalpha transactivation of gene expression||June 11, 2018 22:56|
|Decreased, PPARalpha transactivation of gene expression leads to Fatty Acid Beta Oxidation, Decreased||June 11, 2018 23:05|
|Decreased, PPARalpha transactivation of gene expression leads to Decreased, Ketogenesis (production of ketone bodies)||June 11, 2018 23:21|
|Fatty Acid Beta Oxidation, Decreased leads to Decreased, Ketogenesis (production of ketone bodies)||June 11, 2018 23:14|
|Decreased, Ketogenesis (production of ketone bodies) leads to Not Increased, Circulating Ketone Bodies||June 11, 2018 23:25|
|Not Increased, Circulating Ketone Bodies leads to Increased, Catabolism of Muscle Protein||June 11, 2018 23:30|
|Increased, Catabolism of Muscle Protein leads to Decreased, Body Weight||June 11, 2018 23:33|
|GW6471||January 30, 2017 16:02|
|Nitrotoluenes (hypothesized binding)||January 30, 2017 16:26|
|PPARalpha antagonists||June 02, 2017 14:46|
The present AOP describes antagonistic chemical binding to the peroxisome proliferator-activated receptor α (PPARα) resulting in preferential binding a co-repressor to the overall PPARα signaling complex causing a chain of events that includes: antagonism of PPARα nuclear signaling, decreased transcriptional expression of PPARα-regulated genes that support energy metabolism, and inhibited metabolic energy production culminating with starvation-like weight loss. The AOP is likely to be synergized during fasting, starvation or malnutrition events. The MIE for this AOP involves antagonistic PPARα binding. The antagonist-binding to the PPARα regulatory complex causes the KE1, stabilization of co-repressor (SMRT or N-CoR) to PPARalpha ligand binding domain suppressing PPARα nuclear signaling (Nagy et al 1999, Xu et al 2002). PPARα is a transcriptional regulator for a variety of genes that facilitate systemic energy homeostasis (Mirza et al 2019, Kersten 2014, Evans et al 2004, Desvergne and Wahli 1999). As a result of the MIE and then KE1, the KE2 occurs where PPARalpha transactivation is inhibited for genes involved in the next 2 key events of the AOP: (KE3) decreased fatty acid beta oxidation (Desvergne and Wahili 1999, Kersten 2014, Dreyer et al 1992, Lazarow 1978, Brandt et al 1998; Mascaro et al 1998, Aoyama et al 1998, Gulick et al 1994, Sanderson et al 2008) and (KE4) decreased ketogenesis (Cahil 2006, Kersten et al 2014, Sengupta et al 2010, Desvergne and Wahli 1999). The KE3 results in decreased catabolism of very long chain fatty acids in peroxisomes and reduced catabolism of long, medium and short chain fatty acids in mitochondria reducing acetyl-CoA availability for use in oxidative phosphorylation-based ATP production (Evans et al 2004). KE2 (and also potentially KE3) can drive KE4 resulting in decreased potential to repackage energy substrates as ketone bodies to support systemic energy demands during periods where the systemic energy budget is negative (Badman et al 2007, Potthoff 2009; Muoio et al 2002). The KE5, no change or a decrease in circulating ketone bodies becomes critical during cellular energy deficit conditions, a state where ketogenesis is typically induced to increase circulating ketone bodies providing metabolic fuel to sustain energy homeostasis (Cahill 2006). Physiological studies of the progression of human starvation have demonstrated the critical importance of ketogenesis, especially production of β-hydroxybutyrate, for meeting systemic energy demands by supplementing glucose to sustain the energy requirements of the brain (Cahill 2006, Owen et al 2005). PPARα knock can inhibit ketogenesis from fatty acid substrates in fasted mice reducing β-hydroxybutyrate production causing hypoketonemia (Badman et al 2007, Le May et al 2000, Muoio et al 2002). Sustained negative energy budgets lead to KE6, an increase in muscle protein catabolism, with glutamine and alanine recycled for gluconeogenesis (Felig et al 1970A, Kashiwaya et al 1994). If ketogenesis from fatty acid substrates fails to meet cellular energy needs, gluconeogenesis from alternative substrates becomes necessary including (KE 6) muscle protein catabolism in situ supporting local muscle function and releasing glutamine (Marliss et al 1971) and alanine (Felig et al 1970A) for gluconeogenesis in kidney and liver to sustain systemic energy needs (Goodman et al 1966, Kashiwaya et al 1994, Cahill 2006). Finally, the AO of body-weight loss occurs, which within the context of dynamic energy budget theory, decreases energy allocations to organismal maturation and reproduction (Nisbet et al 2000) and has been demonstrated to negatively affect ecological fitness (Martin et al 1987).
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||998||Binding of antagonist, PPAR alpha||Binding of antagonist, PPAR alpha|
|2||KE||1000||stabilization, PPAR alpha co-repressor||stabilization, PPAR alpha co-repressor|
|3||KE||858||Decreased, PPARalpha transactivation of gene expression||Decreased, PPARalpha transactivation of gene expression|
|4||KE||1528||Fatty Acid Beta Oxidation, Decreased||Fatty Acid Beta Oxidation, Decreased|
|5||KE||861||Decreased, Ketogenesis (production of ketone bodies)||Decreased, Ketogenesis (production of ketone bodies)|
|6||KE||862||Not Increased, Circulating Ketone Bodies||Not Increased, Circulating Ketone Bodies|
|7||KE||863||Increased, Catabolism of Muscle Protein||Increased, Catabolism of Muscle Protein|
|8||AO||864||Decreased, Body Weight||Decreased, Body Weight|
Relationships Between Two Key Events (Including MIEs and AOs)
|Binding of antagonist, PPAR alpha leads to stabilization, PPAR alpha co-repressor||non-adjacent||High||Moderate|
|Not Increased, Circulating Ketone Bodies leads to Increased, Catabolism of Muscle Protein||non-adjacent||Moderate||Low|
Life Stage Applicability
Overall Assessment of the AOP
The majority of the evidence described in this AOP are derived for either human (mostly in vitro) or mice (in vivo and in vitro) studies. There are recognized differences between mouse and human PPARα signaling and responses from the literature, however, for our specific KEs, the responses among species are relatively well conserved. Therefore, we have reasonable confidence that the AOP provides reliable confidence for human health assessment. The AOP also has the potential to support ecotoxicological assessment if there is reasonable confidence that the KEs are conserved in the species of interest. The risk for this AOP is expected to be exacerbated during fasting, starvation and/or sub-optimal nutrition where interference with PPARα signaling is likely to contribute synergistically toward decreased exercise performance in the short-term and drive body-weight loss in long-term exposures. The molecular responses from the MIE through KE4 are very well characterized in the literature for human and mouse. KE5 and KE6 have fairly strong support from the literature, however the KER between them, especially stemming back to the MIE remains the largest data gap within the AOP. Finally, the connection between KE6 and the AO is intuitive and well established in the literature. Overall, the AOP is biologically plausible with logical order where AO is likely to be exacerbated when nutrition is suboptimal.
Regarding the use of the AOP for chemical safety assessment, the AOP should have relevance for any chemical observed to inhibit PPARα signaling. Additionally, the manifestation and severity of the AO is expected to occur predominantly in chronic exposures, especially in nutritionally stressed populations. There is much left to learn about what chemical structures are likely to antagonistically bind to PPARα before quantitative structure-activity relationships (QSARs) can be developed to predict binding / antagonistic effects.
Domain of Applicability
The majority of the evidence described in this AOP are derived for either human (mostly in vitro) or mice (in vivo and in vitro) studies. There are recognized differences between mouse and human PPARα signaling and responses from the literature, however, for our specific KEs, the responses among species are relatively well conserved. Therefore, we have reasonable confidence that the AOP provides reliable confidence for human health assessment. The AOP also has the potential to support ecotoxicological assessment if there is reasonable confidence that the KEs are conserved in the species of interest.
Essentiality of the Key Events
Rationale for essentiality calls:
- MIE: PPARα, Binding of antagonist: Regarding the present MIE, molecules can bind to the PPARα regulatory complex affecting the binding of co-activators and co-repressors. Specifically designed molecules such as the PPARα antagonists GW6471 can bind to PPARα selectively recruiting binding of co-repressors to the PPARα nuclear signaling complex (Xu et al 2002).
- Key Event 1: PPAR alpha co-repressor, Increased - The binding of co-repressors to the PPARα signaling complex suppresses nuclear signaling and thus downstream transcription of PPARα-regulated genes (Liu et al 2008). GW6471 binding to the co-repressor is reversible thus allowing the co-repressor to leave the ligand binding domain of PPARα, restoring normal function (Xu et al 2002).
- Key Event 2: PPARalpha transactivation of gene expression, Decreased - As described in a variety of reviews, PPARalpha represents a master regulator of energy metabolism which specifically promotes fatty oxidation for energy production & distribution (Evans et al 2004, Kersten 2014, Lefebvre et al 2006, Desvergne and Wahili 1999, Mirza et al 2019). Both PPARalpha knock outs and PPARalpha antagonism decreased transcriptional expression of gene targets involved in peroxisomal fatty acid beta oxidation (Kersten et al 1999, Desvergne and Wahili 1999, Janssen et al 2015), mitochondrial fatty acid beta oxidation (Brandt et al 1998; Mascaro et al 1998, Kersten 2014), and ketogenesis (Sengupta et al 2010, Desvergne and Wahli 1999, Kersten 2014).
- Key Event 3: Fatty Acid Beta Oxidation, Decreased – This key event is essential for deriving metabolic energy from fatty acid substrates thus supporting a large component of overall organismal energy demands (Evans et al 2004, Kersten 2014, Desvergne and Wahili 1999). Very long chain fatty acids (>C20) are metabolized in the peroxisome and short, medium and long chain fatty acids (<C20) are catabolized by mitochondrial beta-oxidation. PPARalpha regulates nearly every enzymatic step in the uptake as well as the oxidative breakdown of acyl-CoAs to acetyl-CoA (Kersten 2014). The acetyl-CoA monomers serve as fundamental units for metabolic energy production (ATP) via the citric acid cycle followed by electron-transport chain mediated oxidative phosphorylation (Nelson and Cox, 2000A) as well as serve as the fundamental units for energy storage via gluconeogenesis (Nelson and Cox, 2000B) and lipogenesis (Nelson and Cox, 2000C). PPARalpha knockout studies have demonstrated impaired mitochondrial fatty acid oxidation leading to fatty acid accumulation in the liver (Badmann et al 2007) as well as an inability to meet systemic energy demands (Kersten et al, 1999).
- Key Event 4: Ketogenesis (production of ketone bodies), decreased - The liver represents a key organ involved in systemic energy distribution given its ability to synthesize glucose (an ability shared only with the kidney, Gerich et al 2001) as well as its exclusive role in the generation of ketone bodies (Cahill 2006, Sengupta et al 2010, Kersten 2014). This is especially important for the metabolic energy needs of the brain which can only use glucose and the ketone body, β-hydroxybutyrate for cellular energy production (Cahill 2006, Owen 2005, Kersten 2014). Therefore, ketogenesis is critical to supporting general systemic energy homeostasis in fasting events (Cahill 2006, Evans et al 2004, Sengupta et al 2010). Interference with ketogenesis, for example by PPARα inhibition, has been demonstrated to inhibit β-hydroxybutyrate production (measured in serum) during fasting events in mice (Le May et al 2000, Badman et al 2007, Potthoff 2009, Sengupta et al 2010) and cause hypoketonemia (Muoio et al 2002). The Badman et al (2007) study indicated that metabolism of fatty acid substrates (measured as liver triglycerides) that would otherwise contribute to β-hydroxybutyrate production was additionally inhibited under PPARα knockout.
- Key Event 5: Circulating Ketone Bodies, Not Increased - Physiological studies of the progression of human starvation have identified that the preferred metabolic fuel is glucose in the fed state and progressing through two days of fasting, afterward ketone bodies become increasingly important for meeting energy demands (Cahill 2006, Owen et al 2005). Substrates derived from carbohydrates, fats and protein can contribute to gluconeogenesis (Cahill 2006, Gerich et al 2001) whereas substrates derived from fatty acids are the primary contributors to ketogenesis (Desvergne and Wahli 1999). Cahill (2006) and colleagues have demonstrated the importance of ketone body production, especially β-hydroxybutyrate, for maintaining energy homeostasis during starvation by serving as an alternative substrate to glucose for providing energy to the brain in the starvation state (Cahill 2006). Interference with ketogenesis, for example by PPARα inhibition, has been demonstrated to inhibit β-hydroxybutyrate production (measured in serum) during fasting events in mice (Badman et al 2007, Potthoff 2009). Related to this observation, PPARα-knockout mice reached exhaustion sooner than wild types in an exercise challenge which corresponded with significantly decreased β-hydroxybutyrate in serum indicating hypoketonemia in PPARα-knockout mice versus wild types (Muoio et al 2002). Under normal conditions, activated ketogenesis occurring during fasting events is rapidly deactivated when blood glucose concentrations increase to normal levels and resultant elevated circulating ketone bodies are reduced correspondingly (Cahill 2006).
- Key Event 6: Catabolism of Muscle Protein, Increased - After two to three days of fasting in humans, dietary glucose has been long-since expended and contribution to blood glucose from glycogen metabolism is reduced to zero (Cahill 2006). At this point, about two fifths of fatty acid metabolism in the whole body is dedicated to hepatic ketogenesis, largely in support of the energy demands of the brain, however the brain is still significantly supported by glucose derived from gluconeogenesis (Cahill 2006). As fatty acid stores are depleted, gluconeogenesis from other substrates becomes increasingly important including muscle protein catabolism in situ for supporting muscle function as well as releasing glutamine (Marliss et al 1971) and alanine (Felig et al 1970A) which can be recycled to glucose by gluconeogenesis in the kidney (Goodman et al 1966, Kashiwaya et al 1994, Cahill 2006). In prolonged starvation events, the catabolism of muscle protein for gluconeogenesis in order to support systemic energy needs results in loss of muscle mass which contributes to loss of overall body weight. This loss is rapidly reversible upon input of alternative metabolic fuel for example by nutrient assimilation from feeding.
- Adverse Outcome: Loss of body weight - If caloric intake is less than caloric use over time, an individual will lose body weight. Dynamic energy budget theory has provided useful insights on how organisms take up, assimilate and then allocate energy to various fundamental biological processes including maintenance, growth, development and reproduction (Nisbet et al 2000). Regarding energy allocation, somatic maintenance must first be met before then growth may occur, followed by maturation and then finally, surplus energy is dedicated to reproduction (Nisbet et al 2000). The influence of PPARalpha on systemic energy metabolism and energy homeostasis has been broadly established (see reviews by Kersten 2014, Evans et al 2004, Desvergne and Wahli 1999). PPARalpha has been demonstrated to play a critical role in stimulating fatty acid oxidation and ketogenesis during fasting resulting in increased ketone body levels in plasma (Badman et al 2007, Kersten 2014) a response that is eliminated in PPARalpha knockout mice (Badman et al 2007, Sanderson et al 2010). Kersten et al (1999) and Badman et al (2007) demonstrated that PPARalpha-null mice were unable to actively mobilize fatty acid oxidation, and further, Kersten et al (1999) demonstrated that these mice were unable to meet energy demands during fasting and leading to hypoglycemia, hyperlipidemia, hypoketonemia and fatty liver. Observations from toxicological and toxicogenomic research have implicated nitrotoluenes as potential PPAR antagonists in birds (Rawat et al 2010), rats (Deng et al 2011) and mice (Wilbanks et al 2014), an effect that additionally corresponded with weight loss in rats (Wilbanks et al 2014) and body weight loss, loss of muscle mass and emaciation in birds (Quinn et al 2007). These combined results indicate that inhibition of PPARalpha signaling and the resultant decrease in fatty acid oxidation and ketogenesis can detrimentally impair systemic energy budgets leading to starvation-like effects and resultant weight loss. In the absence of PPARalpha knockout, and upon removal of PPARalpha antagonist dosing, normal bioenergetic physiology can potentially be attained.
Binding of molecules to peroxisome proliferator-activated receptor α (PPARα) can cause either agonistic (i.e. GW409544) or antagonistic (i.e. GW6471) signaling depending on molecular structure (Xu et al 2001, Xu et al 2002). A well described mode of antatonistic binding by GW6471 demonstrates that the molecule can bind to the PPARα ligand binding domain causing conformational changes that induce increased affinity to co-repressors which decrease PPARα nuclear signaling (Xu et al 2002) representing the MIE for this AOP.
The transcription co-repressors, silencing mediator for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR) have been observed to compete with transcriptional co-activators for binding to nuclear receptors (including PPARα) thus suppressing basal transcriptional activity (Nagy et al 1999, Xu et al 2002). Regarding the KE1, the binding of co-repressors such as the SMRT and N-CoR to PPARα is reinforced by the MIE, which blocks the AF-2 helix from adopting the active conformation, as demonstrated in x-ray crystallography results presented in Xu et al (2002). Thus, molecules that bind to PPARα that can enhance co-repressor binding act as PPARα antagonists.
Given that PPARα trans-activation induces catabolism of fatty acids, this signaling pathway has been broadly demonstrated to play a key role in energy homeostasis (Kersten 2014, Evans et al 2004, Desvergne and Wahli 1999). In fact, PPARα regulates expression of genes encoding nearly every enzymatic step of fatty acid catabolism including fatty acid uptake into cells, fatty acid activation to acyl-CoAs, the release of cellular energy from fatty acids through the oxidative breakdown of acyl-CoAs to acetyl-CoA, and in starvation conditions, the repackaging of Acetyl-CoA substrates into ketone bodies (Kersten 2014, Desvergne and Wahli 1999, Evans et al 2004, Sengupta et al 2010).
A large body of research demonstrated that PPARα nuclear signaling directly controls transcriptional expression for genes catalyzing peroxisomal beta-oxidation of very long chain fatty acids (>20C), mitochondrial beta-oxidation of short, medium and long chain fatty acids (<20C), and ketogenesis (as reviewed in Kersten 2014, Evans et al 2004, Desvergne and Wahli 1999, Sanderson et al 2010, McMullen et al 2014, Rakhshandehroo et al 2009).
Peroxisomal beta-oxidation reactions shorten very long chain fatty acids from dietary sources releasing acetyl-CoA subunits (a primary metabolic fuel source) and shortened-chain fatty acids that can subsequently be catabolized by mitochondrial fatty acid beta oxidation reaction (as reviewed in Kersten et al 2014 and Desvergne and Wahli 1999). Fatty acids shortened via peroxisomal beta-oxidation as well as fatty acids released from adipose tissue stores can be catabolized in mitochondrial beta-oxidation reactions to acetyl-CoA, NADH and ATP (Aoyama et al 1998). Within the mitochondria, the acetyl-CoA substrates can be used to maximize ATP production through full substrate oxidation via the citric acid cycle followed by oxidative phosphorylation by the electron transport chain (Nelson and Cox 2000A, Desvergne and Wahli 1999). This demonstrates the importance of PPARα signaling for inducing cellular energy release from fatty acids.
Blocking PPARα signaling has been shown to inhibit expression of transcripts / enzymes involved in both peroxisomal and mitochondrial beta-oxidation causing impaired fatty acid catabolism, fatty acid accumulation in the liver and impaired cellular energy state during fasting events (Badman et al 2007, Kersten et al 1999).
During periods of fasting, acetyl-CoA generated during either peroxisomal or mitochondrial beta-oxidation of fatty acids in the liver can each contribute to ketogenesis (Kersten 2014, Sengupta 2010). The liver represents a key organ involved in systemic energy distribution given its ability to synthesize glucose and catalyze the formation of ketone bodies, especially β-hydroxybutyrate, via ketogenesis (Cahil 2006, Kersten 2014). β-hydroxybutyrate is especially important for the metabolic energy needs of the brain which is unable to utilize fatty acids for cellular energy production (Owen 2005, Kersten 2014) as well as supporting general systemic energy homeostasis in fasting events (Cahil 2006, Evans et al 2004).
Not only does PPARα signaling stimulate the release of cellular energy from fatty acids, it also regulates the transcription of enzymes that catalyze the repackaging of that cellular energy to ketone bodies via ketogenesis (Sengupta et al 2010, Desvergne and Wahli 1999). Inhibition of PPARα signaling has been demonstrated to inhibit transcriptional expression of genes that catalyze ketogenesis as well as ketone body production (Badman et al 2007, Potthoff 2009, Sengupta 2010) affecting circulating levels of ketone bodies for systemic use.
Kersten et al (1999) demonstrated that PPARα is induced in fasted mice mobilizing the oxidation of fatty acids for energy production. In that study, PPARα-null mice did not actively induce fatty acid oxidation or ketogenesis leaving the mice unable to meet energy demands during fasting and leading to hypoglycemia, hyperlipidemia, hypoketonemia and fatty liver. In such energy deficits, muscle protein catabolism is induced where the amino acids glutamine and alanine serve as substrates for gluconeogenesis in the kidney to supplement cellular energy production / distribution (Cahill 2006, Marliss et al 1971, Felig et al 1970A, Goodman et al 1966, Kashiwaya et al 1994).
PPARα knockout resulted in important organism-level responses including quicker onset of exhaustion compared to wild type mice in exercise trials where PPARα-K/Os exhibited hypoketonemia (Muoio et al 2002). Additionally, animals exposed to pollutants (nitrotoluenes) that act as partial PPARα antagonists had decreased exercise endurance (Wilbanks et al 2014), showed body weight loss (Wilbanks et al 2014, Quinn et al 2007) and displayed loss of muscle mass (Quinn et al 2007).
In general, if caloric intake is less than caloric use over time, an individual will lose body weight. This is a basic principle in human dieting as well as an important principle related to individual health and ecological fitness of animal populations.
Dynamic energy budget theory has provided useful insights on how organisms take up, assimilate and then allocate energy to various fundamental biological processes including maintenance, growth, development and reproduction (Nisbet et al 2000). Regarding energy allocation, somatic maintenance must first be met before then growth may occur, followed by maturation and then finally, surplus energy is dedicated to reproduction (Nisbet et al 2000).
As an example of the importance of energy allocation to ecological fitness, a review by Martin et al (1987) demonstrated that energy availability (availability of food) was the predominant limiting factor in reproductive success and survival for both young and parents in a broad life history review for bird species. This is a likely scenario for many organisms.
"'Concordance of dose-response relationships:"'
Dose-response relationships have been developed for GW6471 and the relative binding of PPARα co-repressors and co-activators to the PPARα nuclear signaling complex where the proportion of co-repressors increases dramatically with increasing GW6471 concentration (Xu et al 2002). Correspondingly, the relative activity of PPARα decreased to zero with increasing GW6471 concentrations (Xu et al 2002). Additionally, recent observations of PPARα antagonism by nitrotoluenes have demonstrated dose-response relationships for PPARα nuclear signaling inhibition in human in vitro investigations which corresponded with dose-responsive decreases in transcriptional expression of genes involved in lipid metabolism pathways (Wilbanks et al 2014, Gust et al 2015). These results corresponded with a dose-responsive relationship where increasing nitrotoluene dose caused decreased muscle mass, decreased body weight and increased emaciation in chronic dosing studies (Quinn et al 2007).
"'Temporal concordance among the key events and adverse effect:"'
Co-repressor binding was observed prior to inhibition of PPARα signaling (Xu et al 2002). PPARα knock out nullifies downstream expression of transcripts for genes involved in peroxisomal beta-oxidation of fatty acids, mitochondrial beta-oxidation of fatty acids, and ketogenesis pathways relative to wild types (Kersten et al 2014). Peroxisomal beta-oxidation of very long chain fatty acids into long chain fatty acids occurs prior to import into mitochondria and progression of mitochondrial beta-oxidation (Lazarow 1978, Kersten 2014). Mitochondrial beta-oxidation of long chain fatty acids occurs prior to generation of ketone bodies via ketogenesis (Sengupta et al 2010, Badman et al 2007). Ketogenesis occurs prior to increases in circulating ketone bodies (Sengupta et al 2010, Badman et al 2007, Cahill 2006). Increases in circulating ketone bodies can be observed prior to loss of muscle mass to muscle-protein catabolism given that this linkage is not directly connected. Muscle protein catabolism derives amino acids that are recycled to glucose via renal gluconeogenesis (Goodman et al 1966, Kashiwaya et al 1994, Cahill 2006). Catabolism of muscle protein occurs prior to body weight loss (Quinn et al 2007).
The transcription co-repressors, silencing mediator for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR) competition with transcriptional co-activators for binding to nuclear receptors (including PPARα) has been observed in humans as well as yeast (Nagy et al 1999) suggesting broad taxonomic applicability for this MIE. The evidence of PPARα as a regulator of fatty acid metabolism is well described in the literature (for example, Kersten 2014, Evans 2004, Desvergne and Wahili 1999), and is consistent across many species including human, mouse, rat, Northern bobwhite, fathead minnow and carp (Kersten et al 1999, Kersten 2014, Wintz et al 2006, Gust et al 2015, Deng et al 2011, Wilbanks et al 2014, Xu and Jing, 2012). Inhibition of PPARα via gene knockout or treatment with PPARα antagonist consistently results in deceased fatty acid metabolism with indicators of increased serum triglycerides, fatty livers and steatosis (Kersten 2014, Evans 2004, Desvergne and Wahili 1999, Kersten et al 1999, Wintz et al 2006, Deng et al 2011). Given PPARα’s central role in systemic energy metabolism, studies of PPARα antagonism have shown decreased potential for sustaining energy needs of the organism (Kersten et al 1999) leading to decreased exercise performance (Muoio et al 2002, Wilbanks et al 2014) and weight loss (Wilbanks et al 2014, Quinn et al 2007). Research thus far suggest that the PPARα transcriptional regulation pathway as well as the metabolic pathways for which PPARα acts as a regulator indicates that the progression of key events through to the adverse outcome will tend to be evolutionarily conserved for within mammals and likely across animal phyla.
"'Uncertainties, inconsistencies, and data gaps:"'
A critical data gap regarding this AOP is an absence of studies that have investigated the effects null mutants for ketogenesis on the physiology and individual performance during long term starvation relative to wild type individuals. Additionally, knowledge about feedback mechanisms between ketogenesis vs gluconeogenesis would be beneficial for interpreting systemic energy metabolism. Regarding the antagonistic action of nitrotoluenes on PPARalpha nuclear signaling (Wilbanks et al 2014, Gust et al 2015), receptor-binding assays would be beneficial to determine if this class of chemicals is binding the SMRT and N-CoR co-repressors, similar to the antagonistic action of GW6471 (Xu et al 2002).
Given the complex nature of PPARalpha’s functioning within a multi-subunit transcription factor regulating the transcriptional expression of a multitude of genes that facilitate lipid metabolism, to our knowledge, the relationship between PPARalpha signaling and individual gene expression has not yet been quantitatively modeled. However, the gene regulatory networks structure is well established (KEGG Pathway, map03320) and numerous empirical observations of the positive relationship between PPARalpha signaling with transcript expression and downstream metabolic pathways (Kersten 2014, Desvergne and Wahli 1999), there is opportunity to develop a quantitative gene signaling model for this system. For peroxisomal and mitochondrial fatty acid beta-oxidation pathways and ketogenesis, a variety of enzyme kinetics information is available for modeling (see reviews by Kersten 2014, Desvergne and Wahli 1999) as well as basic knowledge of the reaction stoichiometry of each metabolic reactions that can contribute to metabolic energy substrates for systemic use. Resultant models should be integrated with the work of Kashiwaya et al (1994) who have developed a detailed quantitative model for the metabolic flux of glucose including the influence of ketone bodies and insulin action on the dynamics of glycolysis versus gluconeogenesis. Dynamic energy budget (DEB) models (Nisbet et al 2000) have strong utility for integrating the dynamics of energy input and allocation to organismal processes of importance for characterizing/predicting the condition of the individual (ie. growth and maturation) as well as population-level responses (ie. allocation of energy to reproduction). DEB modeling has great potential for integrating suborganismal processes into individual and population level outcomes (Ananthasubramaniam et al 2015) and could serve to integrate data from dose-responsive relationships among PPARalpha antagonistic nitrotoluenes and fatty acid metabolism, muscle loss and body weight loss (Rawat et al 2010, Deng et al 2011, Wilbanks et al 2014, Quinn et al 2007, Xu and Jin 2012) thus supporting development of a semi-quantitative or quantitative AOP.
Considerations for Potential Applications of the AOP (optional)
The present AOP may require additional conditions to be fully manifested. The risk for this AOP is expected to be exacerbated during fasting, starvation and/or sub-optimal nutrition where interference with PPARα signaling is likely to contribute synergistically toward decreased exercise performance and increased body-weight loss.
Ananthasubramaniam B, McCauley E, Gust KA, Kennedy AJ, Muller EB, Perkins EJ, Nisbet RM: Relating suborganismal processes to ecotoxicological and population level endpoints using a bioenergetic model. Ecol Appl 2015, 25(6):1691-1710.
Aoyama, T., Peters, J.M., Iritani, N., Nakajima, T., Furihata, K., Hashimoto, T., et al., 1998. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). Journal of Biological Chemistry 273:5678e5684.
Badman MK, Pissios P, Kennedy AR, Koukos G, Flier JS, Maratos-Flier E: Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states. Cell metabolism 2007, 5(6):426-437.
Brandt JM, Djouadi F, Kelly DP: Fatty acids activate transcription of the muscle carnitine palmitoyltransferase I gene in cardiac myocytes via the peroxisome proliferator-activated receptor alpha. J Biol Chem 1998, 273(37):23786-23792.
Cahill Jr., G.F., 2006. Fuel metabolism in starvation. Annual Review of Nutrition 26:1e22.
Collier ZA, Gust KA, Gonzalez-Morales B, Linkov I, Perkins EJ (Eligible for publication, pending revisions) A Weight of Evidence Assessment Approach for Adverse Outcome Pathways. Regul Toxicol Pharm.
Deng Y, Johnson DR, Guan X, Ang CY, Ai J, Perkins EJ (2010) In vitro gene regulatory networks predict in vivo function of liver. BMC Systems Biology 4:153.
Deng, Y., Meyer, S. A., Guan, X., Escalon, B. L., Ai, J.,Wilbanks, M.S., Welti, R., Garcia-Reyero, N. and Perkins, E. J. (2011) Analysis of common and specific mechanisms of liver function affected by nitrotoluene compounds. PLoS One 6, e14662.
Desvergne B, Wahli W (1999) Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews 20(5): 649-688.
Evans RM, Barish GD, Wang YX: PPARs and the complex journey to obesity. Nat Med 2004, 10(4):355-361.
Feige, J.N., Gerber, A., Casals-Casas, C., Yang, Q., Winkler, C., Bedu, E., Bueno, M., Gelman, L., Auwerx, J., Gonzalez, F.J., Desvergne, B., 2010. The pollutant diethylhexyl phthalate regulates hepatic energy metabolism via species-specific PPARalpha-dependent mechanisms. Environ. Health Perspect. 118, 234-241.
Felig P, Pozefsky T, Marliss E, Cahill GF, Jr.: Alanine: key role in gluconeogenesis. Science 1970A, 167(3920):1003-1004.
Felig P, Marliss E, Pozefsky T, Cahill GF, Jr.: Amino acid metabolism in the regulation of gluconeogenesis in man. Am J Clin Nutr 1970B, 23(7):986-992.
Gerich JE, Meyer C, Woerle HJ, Stumvoll M: Renal gluconeogenesis: its importance in human glucose homeostasis. Diabetes Care 2001, 24(2):382-391.
Goodman AD, Fuisz RE, Cahill GF: Renal gluconeogenesis in acidosis, alkalosis, and potassium deficiency: its possible role in regulation of renal ammonia production. J Clin Invest 1966, 45(4):612-619.
Gust KA, Nanduri B, Rawat A, Wilbanks MS, Ang CY, Johnson DR, Pendarvis K, Chen X, Quinn Jr. MJ, Johnson MS, Burgess SC, Perkins EJ (2015) Systems Toxicology Identifies Mechanistic Impacts of 2-amino-4,6-dinitrotoluene (2A-DNT) Exposure in Northern Bobwhite. BMC Genomics. In Press.
Ide T, Shimano H, Yoshikawa T, Yahagi N, Amemiya-Kudo M, Matsuzaka T, Nakakuki M, Yatoh S, Iizuka Y, Tomita S, et al (2003) Cross-talk between peroxisome proliferator-activated receptor (PPAR) alpha and liver X receptor (LXR) in nutritional regulation of fatty acid metabolism. II. LXRs suppress lipid degradation gene promoters through inhibition of PPAR signaling. Mol Endocrinol 17(7):1255-1267.
Janssen, A.W., Betzel, B., Stoopen, G., Berends, F.J., Janssen, I.M., Peijnenburg, A.A., Kersten, S., 2015. The impact of PPARalpha activation on whole genome gene expression in human precision cut liver slices. BMC Genomics 16, 760.Kashiwaya Y, Sato K, Tsuchiya N, Thomas S, Fell DA, Veech RL, Passonneau JV: Control of glucose utilization in working perfused rat heart. J Biol Chem 1994, 269(41):25502-25514.
Kersten S. 2014. Integrated physiology and systems biology of PPARalpha. Molecular Metabolism 2014, 3(4):354-371.
Kersten S, Seydoux J, Peters JM, Gonzalez FJ, Desvergne B, Wahli W: Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting. J Clin Invest 1999, 103(11):1489-1498.
Lazarow PB: Rat liver peroxisomes catalyze the beta oxidation of fatty acids. J Biol Chem 1978, 253(5):1522-1528.
Le May et al., 2000. Reduced hepatic fatty acid oxidation in fasting PPARK null mice is due to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression. FEBS Lett. 475: 163-166.
Lefebvre P, Chinetti G, Fruchart J-C, Staels B (2006) Sorting out the roles of PPARα in energy metabolism and vascular homeostasis. Journal of Clinical Investigation, 116(3), 571–580.
Liu, M.H., Li, J., Shen, P., Husna, B., Tai, E.S., Yong, E.L., 2008. A natural polymorphism in peroxisome proliferator-activated receptor-alpha hinge region attenuates transcription due to defective release of nuclear receptor corepressor from chromatin. Mol. Endocrinol. 22, 1078-1092.
Marliss EB, Aoki TT, Pozefsky T, Most AS, Cahill GF: Muscle and splanchnic glutamine and glutamate metabolism in postabsorptive and starved man. J Clin Invest 1971, 50(4):814-817.
Martin TE: Food as a limit on breeding birds: a life-history perspective. Annu Rev Ecol Syst 1987:453-487.
Mascaró C, Acosta E, Ortiz JA, Marrero PF, Hegardt FG, Haro D: Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor. J Biol Chem 1998, 273(15):8560-8563.
McMullen, P.D., Bhattacharya, S., Woods, C.G., Sun, B., Yarborough, K., Ross, S.M., Miller, M.E., McBride, M.T., LeCluyse, E.L., Clewell, R.A., Andersen, M.E., 2014. A map of the PPARalpha transcription regulatory network for primary human hepatocytes. Chem. Biol. Interact. 209, 14-24.
Muoio, D.M., MacLean, P.S., Lang, D.B., Li, S., Houmard, J.A., Way, J.M., Winegar, D.A., Corton, J.C., Dohm, G.L., Kraus, W.E., 2002. Fatty acid homeostasis and induction of lipid regulatory genes in skeletal muscles of peroxisome proliferator-activated receptor (PPAR) alpha knock-out mice. Evidence for compensatory regulation by PPAR delta. J. Biol. Chem. 277, 26089-26097.
Nagy L, Kao H-Y, Love JD, Li C, Banayo E, Gooch JT, Krishna V, Chatterjee K, Evans RM, Schwabe JWR: Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev 1999, 13(24):3209-3216.
Nelson DL, Cox MM 2000A. The Citric Acid Cycle. Lehninger Principles of Biochemistry. 3rd Edition. Worth Publishers. New York, NY. p567-592.
Nelson DL, Cox MM 2000B. Carbohydrate Biosynthesis. Lehninger Principles of Biochemistry. 3rd Edition. Worth Publishers. New York, NY. p722-764.
Nelson DL, Cox MM 2000C. Lipid Biosynthesis. Lehninger Principles of Biochemistry. 3rd Edition. Worth Publishers. New York, NY. p770-814.
Nisbet R, Muller E, Lika K, Kooijman S: From molecules to ecosystems through dynamic energy budget models. J Anim Ecol 2000, 69(6):913-926.
Owen OE: Ketone bodies as a fuel for the brain during starvation. Biochem Mol Biol Educ 2005, 33(4):246-251.
Potthoff MJ, Inagaki T, Satapati S, Ding X, He T, Goetz R, Mohammadi M, Finck BN, Mangelsdorf DJ, Kliewer SA et al: FGF21 induces PGC-1α and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response. Proceedings of the National Academy of Sciences 2009, 106(26):10853-10858.
Quinn MJ Jr, Bazar MA, McFarland CA, Perkins EJ, Gust KA, Gogal Jr RM, Johnson MS (2007) Effects of subchronic exposure to 2,6-dinitrotoluene in the northern bobwhite (Colinus virginianus). Environmental Toxicology and Chemistry 26(10):2202-2207.
Rakhshandehroo, M., Hooiveld, G., Muller, M., Kersten, S., 2009. Comparative analysis of gene regulation by the transcription factor PPARalpha between mouse and human. PLoS One 4, e6796.
Rawat A, Gust KA, Deng Y, Garcia-Reyero N, Quinn MJ, Jr., Johnson MS, Indest KJ, Elasri MO, Perkins EJ (2010) From raw materials to validated system: the construction of a genomic library and microarray to interpret systemic perturbations in Northern bobwhite. Physiological Genomics 42(2):219-235.
Sanderson, L.M., Boekschoten, M.V., Desvergne, B., Muller, M., Kersten, S., 2010. Transcriptional profiling reveals divergent roles of PPARalpha and PPARbeta/delta in regulation of gene expression in mouse liver. Physiological Genomics 41:42e52.
Sengupta S, Peterson TR, Laplante M, Oh S, Sabatini DM: mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. Nature 2010, 468(7327):1100-1104.
Wang YX: PPARs: diverse regulators in energy metabolism and metabolic diseases. Cell Res 2010, 20(2):124-137.
Wilbanks, M., Gust, K.A., Atwa, S., Sunesara, I., Johnson, D., Ang, C.Y., Meyer., S.A., and Perkins, E.J. 2014. Validation of a genomics-based hypothetical adverse outcome pathway: 2,4-dinitrotoluene perturbs PPAR signaling thus impairing energy metabolism and exercise endurance. Toxicological Sciences. 141(1):44-58.
Wintz, H., Yoo, L. J., Loguinov, A., Wu, Y., Steevens, J. A., Holland, R. D., Beger, R. D., Perkins, E. J., Hughes, O. and Vulpe, C. D. (2006) Gene expression profiles in fathead minnow exposed to 2,4-DNT correlation with toxicity in mammals. Toxicol. Sci. 94, 71–82.
Wolfrum C, Borrmann CM, Borchers T, Spener F (2001) Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha - and gamma-mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. Proc Natl Acad Sci USA 98(5):2323-2328.
Xu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL, Oplinger JA, Kliewer SA, Gampe RT, McKee DD et al 2001 Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors. Proceedings of the National Academy of Sciences 98(24):13919-13924.
Xu HE, Stanley TB, Montana VG, Lambert MH, Shearer BG, Cobb JE, McKee DD, Galardi CM, Plunket KD, Nolte RT et al: Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPAR[alpha]. Nature 2002, 415(6873):813-817.
Xu, J. and Jing, N. (2012) Effects of 2,4-dinitrotoluene exposure on enzyme activity, energy reserves and condition factors in common carp (Cyprinus carpio). J. Hazard. Mater. 203–204, 299–307.