Uncoupling Mitochondrial Respiration for Diabesity

Introduction

Adipose tissue has been characterized by color: abundant white fat, which stores calories, and mitochondria-packed brown or beige fat cells that burn energy and produce heat. In humans, until as recently as 2009, it was thought that only babies had deposits of brown fat. At that time, small amounts of brown fat were identified in adults, whereas individuals with lower body mass indexes tended to have more brown fat. Brown fat helps babies—who do not yet have the ability to shiver—to stay warm. In adults exposed to cold temperatures, brown fat may serve as an “internal heating jacket” to keep blood warm as it flows back to the heart and brain from cold extremities. PET-CT scans have since shown that most individuals have small amounts of brown fat in the neck and the shoulders with other deposits in the chest and along the axial skeleton.

Brown and beige fats express uncoupling protein 1 (UCP1) that dissipates stored chemical energy in the form of heat. ^{1 –4} Knockout of the Ucp1 gene ⁵ or genetic ablation of brown or beige cells increases susceptibility of the rodents to diabetes and/or obesity, whereas augmentation of the activity or number of these cells can be protective against metabolic disease and obesity. ^{6 –8}

In a process called oxidative phosphorylation, the electron transport chain pumps protons across the inner mitochondrial membrane into the matrix to generate a proton gradient. This gradient drives a molecular machine, the adenosine triphosphate (ATP) synthase, to form ATP. The UCP1 protein catalyzes a “proton leak” that “uncouples” oxidative respiration resulting in a futile cycle, producing heat but no ATP. ^1,4 Augmentation of UCP1 levels was thought until recently to be the primary mediator of the so-called adaptive thermogenesis. ⁹

However, Kazak et al. described a creatine-driven substrate cycle that enhanced energy expenditure and thermogenesis in beige fat ¹⁰ and now Long et al. describe a unique enzyme (PM20D1) secreted by beige and brown fat cells that catalyzes the formation of N-acyl amino acids. ¹¹ Increasing expression of PM20D1 or administration of N-acyl amino acids directly activates mitochondrial energy expenditure in diet-induced obese (DIO) mice, resulting in significant weight loss.

Novel Enzyme from UCP1+ Adipocytes Promotes Energy Expenditure

Adipose tissue, once thought to be a simple depot for fat storage, is now known to play a key role in energy balance by producing numerous bioactive proteins and “adipokines” such as adiponectin, adipsin, leptin, and so on. ¹² Long et al. sought to identify signature proteins secreted by UCP1+ brown and beige adipocytes. ¹¹ Out of an initial panel of 32 candidates, 1, called PM20D1, contained a signal peptide without any transmembrane domains. ^13,14

To evaluate the potential activity of PM20D1, DIO mice were injected with adeno-associated viral (AAV) vectors expressing PM20D1 and compared with individuals receiving negative control AAV-green fluorescent protein (GFP). ¹⁵ After 40 days, the PM20D1-treated mice gained 9%–10% less body weight. Body composition analysis revealed that the weight difference was due exclusively to a 30% reduction in fat mass with no effect on lean body mass. The PM20D1-treated animals had significantly elevated VO2 and VCO2, indicative of increased energy expenditure with no change in food intake or activity (a potential effect of adrenergic stimulation). ¹¹

PM20D1 Generates N-Lipidated Amino Acid In Vivo

Surprisingly, the increased metabolic rate was not accompanied by an induction of UCP1 nor the recently described futile creatine phosphorylation cycle. ¹⁰ PM20D1 is one of five members of the mammalian M20 peptidase family. Liquid chromatography mass spectrometry profiling of plasma from the mice injected with AAV-GFP or AAV-PM20D1 revealed a novel metabolite at m/z = 428, which corresponded to N-oleoyl phenylalanine (C18:1-Phe). Subsequently, other related N-acyl amino acids were found in plasma at concentrations ranging from 1 to 100 nM. Both short (6 hours or 2 days) and longer (16 days) exposure to cold increased the plasma concentration of PM20D1 as well as several N-acyl amino acid products (e.g., C18:1-Leu/Ile and C18:1-Val). ¹¹

N-Acyl Amino Acids Are Endogenous Uncouplers of Respiration Acting Directly on the Mitochondria

In vitro studies of brown adipose tissue adipocytes revealed that a product of PM20D1, C18:1-Phe, stimulated maximum oxygen consumption rates by almost 200%. This effect was seen with wild type cells as well as UCP1-KO cells, suggesting that UCP1 was not required for this biochemical effect. Structure–activity relationship studies revealed a need for a free amino acid carboxylate. Many, but not all, amino acids were active as conjugates and N-acyl amino acids were much more active that free fatty acids (FFAs) alone. Tetramethylrhodamine methyl (TMRM) ester fluorescence was used to directly measure the mitochondrial membrane potential in live cells. The N-acyl amino acids directly decreased TMRM ester fluorescence by ∼45%. Because the uncoupling effects were observed on isolated mitochondria, efforts were made to identify the mitochondrial target. A UV light-activated conjugate of an N-acyl amino acid was used to identify potential targets in mitochondria. The mitochondrial SLC25A4 and SLC25A5 proteins, also known as ANT1 and ANT2, may be the targets. ¹⁶ Although the consensus function of these proteins is to transport ADP/ATP, they have previously been demonstrated to translocate protons across the inner membrane of the mitochondrion. The N-acyl amino acids generated from the cold-induced PM20D1 enzyme may increase uncoupled respiration by modulating the activity of SCL25-mediated proton flux into the mitochondrial matrix. ¹¹

N-Acyl Amino Acids Improve Glucose Homeostasis and Energy Expenditure in Mice

DIO mice weighing ∼40 g were treated daily for 8 days with vehicle, oleate, or C18:1-Leu (25 mg/kg, i.p.). The N-acyl amino acid-treated mice lost 4.1 g vs. 0.3 and 0.6 g for the vehicle and oleate controls. MRI studies of the mice showed that all of the weight loss was due to a difference in fat mass. The N-acyl amino acid-treated animals exhibited reduced food intake (17%), an improvement in their glucose tolerance and significantly augmented energy expenditure (measured as VO2, VCO2). The respiratory exchange ratio was significantly lower in mice receiving C18:1-Leu, indicating a switch to fats as the metabolic fuel type. Several other N-acyl amino acids (e.g., C18:1-Phe and C20:4-Gly) demonstrated the general property of this class of molecules to uncouple respiration. Finally, plasma liver enzymes, cytokines, and so on were not elevated in the treated animals, demonstrating that the N-acyl amino acids are DIRECTLY augmenting whole body energy expenditure, reducing fat mass, and improving glucose metabolism in the DIO mice by this novel mechanism. ¹¹

Medical Implications

Long et al.'s results suggest a model in which exposure to cold activates UPC1 and activates PM20D1 to catalyze the formation of N-acyl amino acids, which in turn cause proton to leak back across the inner mitochondrial membrane, dissipating the proton gradient, thereby uncoupling oxidative phosphorylation (Fig. 1).

OPEN IN VIEWER

FIG. 1.

N-acyl amino acids uncouple oxidative phosphorylation. Exposure to cold activates UPC1 AND activates PM20D1 to catalyze the formation of N-acyl amino acids, which in turn cause proton to leak back across the inner mitochondrial membrane, dissipating the proton gradient, thereby uncoupling oxidative phosphorylation. Color images available online at www.liebertpub.com/rej

Given our current lack of understanding of PM20D1 regulation and its relevance to human physiology, more detailed information about whether the components of N-acyl amino acids, that is, fatty acids and amino acids, regulate its function will be important.

Interestingly, PM20D1 could not acylate all amino acids to the same extent: phenylalanine is the preferred substrate by far with the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine having some activity. The latter observation has to be reconciled with the known association of BCAAs with type 2 diabetes, ¹⁷ although there is some evidence that it is actually the metabolites of BCAAs that may be more important. Methionine, whose restriction has been reported to confer antiaging effects, ¹⁸ is a weak substrate of PM20D1, and its absence is unlikely to affect the overall formation of other N-acyl amino acids. In any case, it is quite likely that amino acid concentration itself is not rate limiting for PM20D1, which would explain the apparent discordance. Similarly, it is unlikely that fatty acid levels per se, which play a complex role in diabetes and nonalcoholic steatohepatitis (NASH), control the formation of N-acyl amino acids. Of note is that some FFAs are known to act beneficially, including short-chain amino acids ¹⁹ by activation of FFA receptors 2 and 3 ²⁰ and mono-unsaturated (e.g., oleic acid, C18:1), and polyunsaturated long-chain FFAs through FFA1 or other long-chain fatty acids, including α-linolenic acid and docosahexaenoic acid acting through LFA4. ²⁰

What else do we know about N-acyl amino acids themselves? Arachidonoyl serine is known to be vasodilatory and neuroprotective. Arachidonoyl glycine inhibits the sensation of pain, and oleoyl serine reverses bone lost. These diverse activities suggest that N-acyl amino acids have other important biological roles and the specific choice of N-acyl amino acid is probably important. ²¹ It should be noted that Long et al. predominately used oleoyl phenylalanine in their studies, but that exposure of mice to cold for 16 days predominantly induced oleoyl conjugates with alanine, glycine, leucine, isoleucine, and valine. ¹¹

Either PM20D1 or N-acyl amino acids themselves could be developed therapeutically for the treatment of obesity and other obesity-associated disorders such as metabolic syndrome or diabetes. However, much preclinical work needs to be completed before these novel findings can be translated to therapies benefiting humans. Among the questions to be answered are:

• How relevant is this enzyme and metabolic pathway to humans?

○ What are the characteristics of the human enzyme?

The fact that the general findings of Long et al. were performed in two laboratories independently supports the veracity of the claims made about the rodents. What remains in the exciting translation of this work to the specific physiology of brown and beige fats and to the general field of human metabolic disease. It is expected to be safer than earlier attempts to uncouple oxidative phosphorylation. ²²

Conclusion

Studies in mice have identified a novel enzyme pathway that generates N-acyl amino acids that uncouple mitochondrial respiration. Administration of N-acyl amino acids directly or the secreted enzyme, PM20D1, that generates them to DIO mice results in significant weight loss primarily of fat tissue in a short period of time. Discovery of this novel, completely unexpected pathway may provide novel insights into the pathogenesis of diabetes and obesity while being exploited for novel therapies that are much needed for the global pandemic of diabesity.