Tissue specific roles of fatty acid oxidation

Abstract

Mitochondrial long chain fatty acid β-oxidation is a critical central carbon catabolic process. The importance of fatty acid oxidation is made evident by the life-threatening disease associated with diverse inborn errors in the pathway. While inborn errors show multisystemic requirements for fatty acid oxidation, it is not clear from the clinical presentation of these enzyme deficiencies what the tissue specific roles of the pathway are compared to secondary systemic effects. To understand the cell or tissue specific contributions of fatty acid oxidation to systemic physiology, conditional knockouts in mice have been employed to determine the requirements of fatty acid oxidation in disparate cell types. This has produced a host of surprising results that sometimes run counter to the canonical view of this metabolic pathway. The rigor of conditional knockouts has also provided clarity over previous research utilizing cell lines in vitro or small molecule inhibitors with dubious specificity. Here we will summarize current research using mouse models of Carnitine Palmitoyltransferases to determine the tissue specific roles and requirements of long chain mitochondrial fatty acid β-oxidation.

Introduction.

Glucose and fatty acids are essential nutrients that enable cells to meet the energetic demands of diverse biological processes. The breakdown of both glucose and fat directly produces the energetic currency of the cell, ATP. While glucose can be broken down in the absence of oxygen into pyruvate/lactate in the cytoplasm, the complete oxidation of glucose in the mitochondrial tricarboxylic acid (TCA) cycle and subsequent oxidative phosphorylation enables the extraction of substantially more energy. Fatty acids require oxygen for their breakdown in mitochondria and are ~2.25 more energy dense than carbohydrates which make them an efficient long term storage vehicle. In times of high energetic demands or during starvation, catecholamine-induced lipolysis of adipose leads to the liberation of fatty acids and increased fat oxidation to sustain aerobic metabolism in diverse cells throughout the body.

There are at least 12 human genetic disorders that result from mutations in enzymes of the fatty acid oxidation pathway, such as CPT1 and CPT2, the carnitine palmitoyltransferases located on the outer and inner mitochondrial membranes, respectively; ACADM (MCAD), medium chain acyl-coA dehydrogenase; ACADVL (VLCAD), very-long chain acyl-CoA dehydrogenase; and ACAT1, acetoacetyl-coA thiolase.^1,2 Disorders of mitochondrial fatty acid oxidation present in a variety of phenotypes, but most share the common clinical features of acute hypoketotic hypoglycemia, enlargement of the liver and steatosis, hypertrophic cardiomyopathy, and skeletal muscle wasting; in mild cases, these outcomes result in intolerance to fasting and strenuous exercise, while, in fatal cases, they result in coma or death. Disorders of fatty acid oxidation have consequences in multiple organ systems, highlighting its importance^{3 4}.

While inborn errors in the pathway demonstrate the requirement of fatty acid oxidation in health and disease, it is not immediately clear what cells/tissues contribute to these processes. That is, what are the tissue specific functions of fatty acid oxidation and under what conditions is it critical or superfluous? In this review, we summarize the tissue specific requirements and metabolic regulation of fatty acid oxidation and how it contributes to tissue bioenergetics and homeostasis during development and response to various physiological cues.

Liver fatty acid oxidation: Gluconeogenesis and Ketogenesis.

The liver is a crucial organ in maintaining whole-body energy homeostasis. To meet the demands of fluctuations in available nutrients, hepatocytes have an incredible dynamic metabolic capacity that allows them to switch between glucose or fatty acids for energy depending on dietary constraints. Carbohydrate-limited diets, high fat diets, illness, and fasting are examples of conditions that increase the dependence of fatty acids as substrates for hepatic energy production and thus an increased reliance on mitochondrial β-oxidation. The liver has the important function during these times to supply peripheral tissues with alternative energy sources, such as ketone bodies, and also plays a role in maintaining blood glucose levels via supporting de novo glucose synthesis. Mitochondrial fatty acid oxidation is critical for both of these key processes.

To understand the requirement of fatty acid oxidation in the liver, mice were generated with a liver-specific knockout of Carnitine Palmitoyltransferase (Cpt2).^5,6 Cpt2 was chosen because it is obligatory for long chain fatty acid β-oxidation, encoded by a single gene and no other enzyme can substitute for its function. Cpt1, which is the regulated step in mitochondrial fatty acid transport and therefore fatty acid oxidation is encoded by two genes, Cpt1a (liver enriched) and Cpt1b (muscle enriched). Eliminating a single Cpt1 enzyme has been shown to increase the other, complicating phenotypic interpretations^7–9. Fatty acid oxidation deficiency created by crossing Cpt2 floxed mice to Albumin-Cre mice (Cpt2^L−/−) did not affect survival during the perinatal period, despite this period being a time in which a majority of nutritional energy is derived from the lipid-laden milk of the dam and the corresponding importance of β-oxidation during this period.^10,11 Adult Cpt2^L−/− knockout mice do not exhibit alterations in body weight, liver weight, or liver triglyceride levels on a low fat or standard chow-fed diet demonstrating the metabolic flexibility and limited role for fatty acid oxidation of the liver during carbohydrate replete conditions. Surprising, liver-specific Cpt2 knockout mice survived a 24hr fast; however, knockout mice exhibit severe hepatomegaly and lipid accumulation and also have increased serum triglyceride and cholesterol levels, suppressed ketone bodies, and depletion of adipose tissue stores. The complete lack of circulating ketone bodies shows the requirement for fatty acid oxidation to provide the hepatic acetyl-CoA necessary to promote ketogenesis¹². Fasting induces a pro-lipid catabolic gene expression program that is induced not only in the liver, but also the kidney, heart, and skeletal muscle, via PPARα induction and subsequent hepatic production of the pro-catabolic endocrine factors such as fibroblast growth factor 21 and growth differentiation factor 15.

Given the robust suppression of ketone bodies, and the requirement of fatty acid oxidation for gluconeogenesis, one would expect Cpt2^L−/− knockout mice to become hypoglycemic following a fast. Yet, Cpt2^L−/− knockout mice maintain blood glucose levels comparable to their littermate controls following a 24hr fast. This is surprising given the hypoglycemia seen in patients with inborn errors in fatty acid oxidation and the purported dominance of gluconeogenesis by the liver. Indeed, fatty acid oxidation is required for the liver to perform gluconeogenesis^5,6. However, the kidney and proximal gut can contribute to gluconeogenesis and is consistent with other liver specific models of deficient gluconeogenesis (Pcx^L−/−, Pepck^L−/−, G6pase^L−/−) that have little or no effect on systemic glucose levels after fasting or starvation^13–16. However, under a carbohydrate-limited ketogenic diet, liver-specific Cpt2 knockout mice fail to thrive and become moribund after a week similar to Pcx^L−/− knockout mice^5,13. While the kidney can compensate for a short term fast, it seems as though long-term glucose control requires the liver.

Cpt2^L−/− knockout mice have a genetically imposed deficit in energy expenditure. Therefore, one might imagine that feeding a high fat diet to mice with defective hepatic fatty acid oxidation would result in a pro-obesity phenotype. However, Cpt2^L−/− mice exhibit a strong resistance to body weight gain and glucose intolerance^6,17. The resistance obesity is mediated by an increase in energy expenditure and can be reversed by housing the mice at thermoneutrality to lower their requirement for energy expenditure. The increase in energy expenditure is accompanied by increases in hepatokines known to increase energy expenditure. Therefore, the phenotype of mice with a loss in long chain mitochondrial beta-oxidation does not comport with our textbook understanding of this central metabolic pathway.

Brain fatty acid oxidation: Astrocyte enriched metabolism.

Bioenergetically, the brain relies most heavily on glucose under most fed state conditions but can somewhat switch to ketone bodies as alternative oxidative substrates during fasting.¹⁸ The importance of mitochondrial fatty acid oxidation in the brain is evidenced by the neurological dysfunction observed in some cases of inborn errors in fatty acid oxidation.^1,2,19 However, it is somewhat unclear if the effects on the nervous system are due to autonomous or nonautonomous roles of fatty acid oxidation. In principle, neurons do not have a robust capacity to perform long-chain fatty acid oxidation, as they express low levels of Cpt1 isoenzymes A and B. The neuron-specific isozyme Cpt1c does not have carnitine acyltransferase activity but likely plays a unique role in neurons.^20–23. Nevertheless, in vitro and in vivo studies using radiolabeled fatty acids and genetically manipulated mouse models indicate that glial cells, neurons, and, most prominently, astrocytes can perform β-oxidation.^24–31 A classic yet overinterpreted study is often cited to conclude that the brain cannot oxidize fatty acids³². However, these studies do not have the rigor to show metabolic requirements only broad capacities. There are circumstances that elicit strong indicators of central fatty acid oxidation. After injury, the brain appears to have increased propensity to perform mitochondrial fatty acid oxidation to meet the increased energetic demand for cellular biogenesis.^33–35

Mouse models that either directly target the pathway or indirectly modulate fatty acid flux have illuminated the role of fatty acid oxidation across the entire brain and in certain cell-types. A mouse model that lacks the ability to perform fatty acid oxidation in the central nervous system via a pan-brain knockout of Cpt2 demonstrated that loss of fatty acid oxidation in the brain results in accumulation of long chain acyl-carnitines, as well as changes in the abundances of other metabolites, such as intermediates of the coenzyme-A biosynthetic pathway, several polyunsaturated fatty acids, short chain acyl-carnitines, and N-acetyl amino acids, thus indicating that the mammalian brain oxidizes fats under basal conditions.³⁶ As long chain acyl-carnitines can be incorporated into phospholipids, the Cpt2 knockout hippocampus also demonstrated an increase in sphingomyelin abundance compared to control, indicating a role for fatty acid oxidation in maintaining brain phospholipid balance. These metabolic findings were correlated with very subtle changes in behavior; as the mice were 9–12 weeks old at the time of the study, it would be interesting to investigate if there would be more changes in the brain metabolome and behavior in aged Cpt2 knockout mice.

Loss of the rate-limiting enzyme in fatty acid oxidation, Cpt1a, specifically in astrocytes is observed to divert the fate of pyruvate from lactate to mitochondrial respiration, decrease electron transport chain supercomplex formation, decrease reactive oxygen species formation, and result in cognitive impairment due to impaired astrocyte-neuron signaling.³⁰ Metabolomics on astrocyte-specific Cpt1a brains also revealed an accumulation of palmitoyl-carnitine compared to wildtype controls. This finding is somewhat unexpected, as Cpt1a is responsible for the production of acyl-carnitines and could suggest that the brain is scavenging exogenous sources of acyl-carnitines or cell types, other cell types in the brain are compensating for the astrocyte-specific loss of acyl-carnitine production, or there is an undetected upregulation of Cpt1b in astrocytes that is functional, amongst other possibilities. In summary, despite the complexity of the metabolic preferences of the different cell types of the brain and the incomplete knowledge we have on the direct effects of fatty acid metabolism on all cell types, it generally appears that mitochondrial β-oxidation contributes to maintaining the brain metabolome, redox homeostasis, and intercellular communication, thus playing a neuroprotective role even though the brain’s relative capacity for fatty acid oxidation is low.

Cardiac fatty acid oxidation: Irreplaceable bioenergetic function.

During the transition from the neonatal to postnatal period, there is a marked shift from glucose utilization to fatty acid metabolism in cardiomyocytes. Gene expression analysis of post-natal sheep and rodent hearts demonstrate that this shift is correlated with progressive expression of several genes involved in lipid metabolism throughout the perinatal period, such as the fatty acid translocase (CD36), acyl-CoA synthetase long-chain 1 (ACSL1), CPT1, hydroxyl-acyl dehydrogenase (HADH), acetyl-CoA acetyltransferase (ACAT1), isocitrate dehydrogenase (IDH), and glycerol phosphate acyltransferase (GPAT), although oxidative metabolism appears to be functionally highest immediately after birth.^37,38 In adults, fatty acid oxidation is estimated to contribute to ~85% of total ATP produced in the human heart.³⁹ Both patients with inherited disorders of fatty acid oxidation and knockout mouse models of the enzymes in the pathway—such as LCAD, CPT1B, CPT2, and ACSL1—exhibit cardiac hypertrophy and overall impaired left ventricular performance and cardiac output, thus highlighting the importance of β-oxidation in preserving heart function.^1,2,40–43

On the metabolic level, a lack of fatty acid oxidation in the hearts of fasted whole-body LCAD knockout mice is correlated with an energetic deficit created by increased reliance on both glucose oxidation and also cardiac anaplerosis, a requirement that cannot not be met due to impaired amino acid mobilization into liver de novo glucose production.^44,45 Both LCAD knockout mice and conditional heart and muscle specific CPT2 knockout mice exhibited hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) pathway in the heart, yet inhibition of the pathway via rapamycin treatment fails to rescue hypoglycemia in LCAD knockouts or cardiac defects in CPT2 knockouts, indicating the requirement of fatty acid oxidation in maintaining cardiomyocyte structure and performance.^41,44 Intriguingly, the failure to activate fatty acids to CoA via knockout of ACSL1 also results impaired fatty acid oxidation, increased glucose oxidation, and hyperactivation of mTORC1, but the cardiac hypertrophy is reversed in these animals following rapamycin treatment, leading to the conclusion that mTORC1 activation is responsible for the hypertrophy phenotype.^46,47

Perhaps these observations are related to free carnitine levels, as both LCAD and CPT2 knockout hearts are depleted in free carnitine, while ACSL1 knockout hearts have ~77% increase in free carnitine relative to their respective controls.^41,43,48,49 Carnitine availability appears to be particularly important in the context of cardiac energetics. Supplementation of L-carnitine alone in LCAD knockout mice is sufficient to beneficially raise free intracellular carnitine levels and reverse triglyceride accumulation in the heart without invoking accumulation of long-chain acyl-carnitines.⁵⁰ Consistent with this observation, low-fat diet fed heart-specific knockout mice of the mitochondrial pyruvate carrier (MPC2) also exhibit cardiac hypertrophy, depletion of free carnitine, and mTORC1 activation.⁵¹ While the primary metabolic defect in this mouse model is in glucose oxidation, it was found that treating MPC2-knockout mice with a ketogenic diet reversed the cardiac hypertrophy, normalized free carnitine levels, and suppressed mTORC1 activation, suggesting that the ability to stimulate fatty acid oxidation plays a protective role in the prevention of cardiac dysfunction.

It is expected that one way to uncouple fatty acid oxidation from the dependency of the carnitine shuttling system is to supplement the diet with a medium chain fatty acid that is oxidized by β-oxidation but does not require the carnitine to enter the mitochondria. This appears to occur in the liver, as octanoate (C8:0) rescues oxidation in CPT2-knockout livers; however, only octanoyl-carnitine rescues mitochondrial oxidation in CPT2-deficient hearts and skeletal muscle.⁴⁸ These data indicate that long chain mitochondrial fatty acid oxidation is required for energizing the heart and regulating cardiac structure.

Recently, several studies have begun to shine light on the signaling role of fatty acid metabolism in the heart beyond bioenergetics. The absence of long chain fatty acid oxidation leads to uncharging of tRNAs, activation of the integrated stress response, and induction of activating transcription factor 4 (ATF4) mRNA and activity in LCAD knockout hearts, possibly contributing to the cardiac hypertrophy phenotype.⁵² While inhibition of CPT1B during development leads to similar detrimental heart defects observed in LCAD and CPT2 knockout hearts, another study found that ablation of fatty acid oxidation by knocking out CPT1B in adult mice specifically after ischemia-reperfusion injury promotes cardiomyocyte proliferation and heart regeneration.⁵³ Mechanistically, loss of fatty acid oxidation results in an accumulation of the TCA cycle intermediate and cytosolic metabolite α-ketoglutarate, also an essential cofactor for histone demethylases, leading to reduced H3K4me3 via activation of the demethylase KDM5 and specific induction of cardiac proliferation genes. The potential expansion and regeneration of adult cardiomyocytes by the inhibition of fatty acid oxidation requires more research⁵⁴.

Skeletal muscle fatty acid oxidation: Upending insulin resistance.

Dysfunctional skeletal muscle fatty acid oxidation has been implicated in the development of insulin resistance and diet-induced obesity.^9,55–58 The skeletal muscle specific loss of CPT1B or CPT2 have been useful in illustrating the role of fatty acid oxidation in muscle and elucidating the roles of long chain acyl-CoA and acylcarnitine accumulation in skeletal muscle. Indeed, both models exhibit resistance to high fat diet-induced weight gain and improved glucose tolerance.^9,55 This was against the expectation that impaired muscle fatty acid oxidation was responsible for insulin resistance. However, there are some key phenotype differences that distinguish the two models on the metabolic and physiological level that can be attributed to the accumulation of acyl-CoAs or acyl-carnitines in the CPT1B and CPT2 models, respectively. Accumulation of acyl-CoAs in the CPT1B model are funneled into triglyceride storage and results in cellular lipid droplets.^9,56 In the CPT2 model, the accumulation of acyl-carnitine in the muscle does not get converted into complex lipids for triglyceride storage.⁵⁵ A paracrine and endocrine effect mediated by muscle fibroblast growth factor 21 (FGF21) and white adipose tissue browning is observed in CPT1B deficiency,⁵⁸ but not in CPT2 deficiency, in which the myomitokine growth differentiation factor 15 (GDF15) is instead upregulated and there are no effects on adipose tissue.⁵⁵ Relative accumulation of acyl-CoAs and acylcarnitines may exert different effects on gene regulation and protein function, leading to the inconsistencies in the phenotypes of these two models. Regardless, the data thus far does not support a causative role for fatty acid oxidation in insulin resistance.

While these studies broadly assessed the tissue-specific and systemic impact of β-oxidation deficiency in skeletal muscle, skeletal muscle in mammals is comprised of a heterogeneous assortment of fibers that exhibit distinct metabolic characteristics.⁵⁹ In general, fibers are classified as type 1 or “slow-twitch”, which are highly oxidative and mitochondria-rich, and type 2 or “fast-twitch”, which exhibit more glycolytic metabolism. At least in rodents, type 2 fibers can be further divided into A, X, and B in order of increasing dependence on glycolysis. The composition of an individual’s fiber types within the skeletal muscle is a determinant of the individual’s tolerance to perform aerobic or anaerobic exercise, with slow-twitch muscle having an increased capacity for oxidative metabolism to support endurance exercise and impart fatigue resistance.^59,60 Indeed, muscles with higher fatty acid oxidative potential are better equipped to respond to increasing bioenergetic demands.⁶¹ As such, fat oxidation contributes to skeletal muscle ATP production during exercise, and inborn errors in β-oxidation can lead to exercise intolerance in some individuals.¹ Additionally, a marked decrease in tolerance to exercise and resistance to fatigue in skeletal muscle is observed when there is a metabolic shift in oxidative to glycolytic metabolism, such as is seen in patients with Friedreich’s ataxia (FRDA, OMIM #229300), and a proteomics approach identified a corresponding shift in identity from type 1 oxidative to type 2 glycolytic fibers as classified by myosin heavy chain isoforms that could explain some of the myopathy attributes to the disorder.⁶²

Mouse models of impaired skeletal muscle fatty acid uptake and oxidation result exhibit decreased physical activity and endurance exercise tolerance,^9,55,57,63 but also intriguingly uncouple metabolism and fiber type.^61,64 The loss of CPT2 elicits a shift in oxidative to glycolytic metabolic program in fibers without impairing mitochondrial function and without inducing fiber type switching as defined by myosin heavy chain isoform composition.⁶¹ Conversely, expression of a mutant form of CPT1B that is insensitive to malonyl-CoA, its endogenous inhibitor, increases the fatty acid oxidative capacity, muscle size, and resistance to muscle fatigue; while the myosin heavy chain isoforms corresponding to oxidative fibers are increased in the overexpressing muscles on the transcriptional level, immunohistochemistry reveals little changes in fiber types.⁶⁴ These observations lead to further questions about the “mismatch” between fiber oxidative potential and the current defining factor of fiber type, myosin heavy chain isoform, that remain to be addressed in the context of muscle physiology.

Despite an increase in mitochondrial biogenesis and the ability to maintain ATP production due to compensatory mitochondrial oxidation of alternative non-fatty acid substrates, the CPT2-deficient muscle exhibits decreased ability to meet bioenergetic demands both in isolated mitochondria and as assessed by treadmill running.^61,65 The results that the decrease in soleus performance in CPT2 knockout muscle is not due to impaired mitochondrial energetics is surprising, as bioenergetic insufficiency has long since been presumed as the cause of skeletal muscle weakness in human patients with CPT2 deficiency. ex vivo analysis of CPT2-deficient glycolytic fibers from the extensor digitorum longus (EDL) and oxidative fibers from the soleus revealed that muscle force production and contractile capacity was specifically impacted in the soleus muscle.⁶⁵ The defects in the structure and contractile ability of soleus muscle in mice lacking CPT2 are driven by downregulation of dystrophin-related genes and contraction-related proteins, leading to functional defects. Furthermore, accumulation of acylcarnitines and decreased cytosolic clearance of calcium into the sarcoplasmic reticulum are observed in CPT2 knockout soleus muscle. Mechanistically, long-chain acylcarnitine exposure to isolated sarcoplasmic reticulum inhibited calcium uptake, therefore demonstrating for the first time a link between long-chain acylcarnitines and calcium dynamics. These results suggest that muscular defects in CPT2-deficient patients could be due to long-chain acylcarnitine inhibiting calcium uptake by the sarcoplasmic reticulum and thus muscle contraction and exercise intolerance. Furthermore, as the soleus muscle can overcome the energetic deficit in the face of loss of fatty acid oxidation, the primary role of fatty acid oxidation appears to be emerging as a regulator of calcium homeostasis and maintaining muscle contractile ability.

Adipose tissue fatty acid oxidation: Fueling the fire.

It is well-established that adipose tissue in mammals plays many endocrine and metabolic roles that are important in maintaining physiological homeostasis. In the most general sense, there are two distinct types of adipose tissue: white adipose tissue (WAT), which is comprised of adipocytes that are unilocular and is the major depot of triglyceride accumulation, and brown adipose tissue (BAT), which is comprised of adipocytes that do not store fat but instead are packed with mitochondria that use fat to fuel thermogenesis. Another type of adipocytes, referred to as beige adipocytes, develop within WAT depots in response to thermogenic stimuli such as cold.⁶⁶ Mice with loss of function or knockout mutations in enzymes in the fatty acid oxidation pathway, including ACSL, CPT2, ACADL, ACADM and ACADS (short chain acyl-coA dehydrogenase), have deficiencies in nonshivering thermogenesis following cold exposure similarly to knockouts of UCP-1, a mitochondrial inner membrane protein required for the uncoupling of oxidative phosphorylation and adipocyte browning, thus highlighting the importance of β-oxidation in the process.^67–71 Paradoxically, loss of fatty acid synthase in adipose tissue results in browning of WAT despite an increase in the inhibitor of fatty acid oxidation, malonyl-CoA, and its carnitine derivative, perhaps indicating that fatty acid oxidation in brown adipocytes is refractory to malonyl-CoA.⁷²

Both ACSL and CPT2 knockout BAT have increased lipid deposition after a cold challenge, but, unlike in ACSL knockouts, the loss of fatty acid oxidation via CPT2 prevents the induction of BAT thermogenic genes following a cold challenge or β3-adregenergic agonism, such as UCP-1, DIO2, and PGC1α, perhaps implicating a role for accumulation of acylcarnitines as a feedback inhibitory mechanism on fatty acid oxidation at the transcriptional level.^69,71 Both ACSL and CPT2 knockout mice exhibit diet-dependent changes in white adipose depots without alterations of weight gain, likely reflecting an increase in energy expenditure in other tissues. CPT2 knockout WAT is protected from high fat diet-induced oxidative stress and inflammation but the mice remain insulin insensitive, indicating that adipose tissue fatty acid oxidation is not a major contributor to insulin resistance following high fat diet.⁷¹ Intriguingly, the loss of CPT2 in adipose tissue does not have an effect on body weight, glucose tolerance, or energetic expenditure at thermoneutrality on or off a high fat diet either, despite a complete phenotypic loss of BAT, suggesting that dysregulated bioenergetics of BAT mediated by loss of fatty acid oxidation alone is not sufficient to drive an obesogenic phenotype as might have been predicted.⁷³ Chronic activation of thermogenic pathways using the agonists CL-316243 and GC-1 also results in deformation of BAT without inducing compensatory browning of WAT.⁷⁴ Additionally, CPT2 mice housed under thermoneutral conditions or chronically treated with agonists of thermogenesis causes a BAT-specific increase in mitochondrial DNA response and innate immune system priming in the former case and macrophage infiltration and fibrosis in the latter case.^73,74 Therefore, beyond being important for fulfilling the bioenergetic needs of thermogenesis, these data indicate that fatty acid oxidation is required for maintenance and survival of brown adipocytes and browning of WAT under metabolic stress.

Beyond canonical roles.

Most cells with mitochondria can oxidize fatty acids if given the opportunity and appropriate physiological setting. Therefore, it is not surprising that diverse tissues have roles and requirements for mitochondrial fatty acid oxidation. Kidney, gut, and endothelium have all been the subject of investigation^75–79. An exciting area of recent study is the role of fatty acid oxidation in the immune system. Several cell types of the immune system acutely alter their mitochondrial metabolism in response to infection or inflammation including T-cells, B-cells and macrophages. It is not immediately clear why these cells pursue such large differences in their metabolism as they need to perform their functions within unique environmental contexts. One popular hypothesis has been that fatty acid oxidation is not only providing the energy required to affect immune cell survival but also instruct their cell fate decisions. This provided an exciting new avenue of study. Much of this work was based on the effect of a popular and often cited specific inhibitor of CPT1 and therefore fatty acid oxidation, etomoxir. However, based on the genetic dissection of fatty acid oxidation in immune cells, it became apparent that etomoxir was not the specific inhibitor the field was lead to believe^80–85. In fact, etomoxir binds promiscuously to diverse fatty acid binding proteins and is particularly efficacious towards binding peroxisomal proteins rather than proteins in mitochondrial fatty acid metabolism⁸⁶. Moving forward, there have not been small molecules identified that are both efficacious and specific to differentiate mitochondrial fatty acid oxidation from other pathways, limiting their utility. Genetic dissection of the pathway remains the most appropriate and rigorous method to assign the roles and requirements of fatty acid oxidation throughout our diverse cells and tissues.