Role of the Mitochondrial Pyruvate Carrier in the Occurrence of Metabolic Inflexibility in Drosophila melanogaster Exposed to Dietary Sucrose.

Excess dietary carbohydrates are linked to dysregulation of metabolic pathways converging to mitochondria and metabolic inflexibility. Here, we determined the role of the mitochondrial pyruvate carrier (MPC) in the occurrence of this metabolic inflexibility in wild-type (WT) and MPC1-deficient (MPC1def) flies that were exposed to diets with different sucrose concentrations for 15-25 days (Standard Diet: SD, Medium-Sucrose Diet: MSD, and High-Sucrose Diet: HSD). Our results showed that MPC1def flies had lower mitochondrial respiration rates than WT flies on the SD and MSD. However, when exposed to the HSD, WT flies displayed decreased mitochondrial respiration rates compared to MPC1def flies. WT flies exposed to the HSD also displayed increased proline contribution and slightly decreased MPC1 expression. Surprisingly, when fed the MSD and the HSD, few metabolites were altered in WT flies whereas MPC1def flies display significant accumulation of glycogen, glucose, fructose, lactate, and glycerol. Overall, this suggests that metabolic inflexibility starts to occur in WT flies after 15-25 days of exposure to the HSD whereas the MPC1def flies display metabolic inflexibility independently of the diet provided. This study thus highlights the involvement of MPC as an essential protein in Drosophila to maintain proper metabolic homeostasis during changes in dietary resources.

Metabolic Characterization and Consequences of Mitochondrial Pyruvate Carrier Deficiency in Drosophila melanogaster.

In insect, pyruvate is generally the predominant oxidative substrate for mitochondria. This metabolite is transported inside mitochondria via the mitochondrial pyruvate carrier (MPC), but whether and how this transporter controls mitochondrial oxidative capacities in insects is still relatively unknown. Here, we characterize the importance of pyruvate transport as a metabolic control point for mitochondrial substrate oxidation in two genotypes of an insect model, Drosophila melanogaster, differently expressing MPC1, an essential protein for the MPC function. We evaluated the kinetics of pyruvate oxidation, mitochondrial oxygen consumption, metabolic profile, activities of metabolic enzymes, and climbing abilities of wild-type (WT) flies and flies harboring a deficiency in MPC1 (MPC1def). We hypothesized that MPC1 deficiency would cause a metabolic reprogramming that would favor the oxidation of alternative substrates. Our results show that the MPC1def flies display significantly reduced climbing capacity, pyruvate-induced oxygen consumption, and enzymatic activities of pyruvate kinase, alanine aminotransferase, and citrate synthase. Moreover, increased proline oxidation capacity was detected in MPC1def flies, which was associated with generally lower levels of several metabolites, and particularly those involved in amino acid catabolism such as ornithine, citrulline, and arginosuccinate. This study therefore reveals the flexibility of mitochondrial substrate oxidation allowing Drosophila to maintain cellular homeostasis.

Dynamic mitochondrial responses to a high-fat diet in Drosophila melanogaster.

Mitochondria can utilize different fuels according to physiological and nutritional conditions to promote cellular homeostasis. However, during nutrient overload metabolic inflexibility can occur, resulting in mitochondrial dysfunctions. High-fat diets (HFDs) are usually used to mimic this metabolic inflexibility in different animal models. However, how mitochondria respond to the duration of a HFD exposure is still under debate. In this study, we investigated the dynamic of the mitochondrial and physiological functions in Drosophila melanogaster at several time points following an exposure to a HFD. Our results showed that after two days on the HFD, mitochondrial respiration as well as ATP content of thorax muscles are increased, likely due to the utilization of carbohydrates. However, after four days on the HFD, impairment of mitochondrial respiration at the level of complex I, as well as decreased ATP content were observed. This was associated with an increased contribution of complex II and, most notably of the mitochondrial glycerol-3-phosphate dehydrogenase (mG3PDH) to mitochondrial respiration. We suggest that this increased mG3PDH capacity reflects the occurrence of metabolic inflexibility, leading to a loss of homeostasis and alteration of the cellular redox status, which results in senescence characterized by decreased climbing ability and premature death.

Omega-3 Monoacylglyceride Effects on Longevity, Mitochondrial Metabolism and Oxidative Stress: Insights from Drosophila melanogaster.

During the last decade, essential polyunsaturated fatty acids (PUFAs) such as eicosatetraenoic acid (EPA) and docosahexaenoic acid (DHA) derived from marine sources have been investigated as nonpharmacological dietary supplements to improve different pathological conditions, as well as aging. The aim of this study was to determine the effects of dietary n-3 PUFA monoacylglycerides (MAG, both EPA and DHA) on the mitochondrial metabolism and oxidative stress of a short-lifespan model, Drosophila melanogaster, sampled at five different ages. Our results showed that diets supplemented with MAG-EPA and MAG-DHA increased median lifespan by 14.6% and decreased mitochondrial proton leak resulting in an increase of mitochondrial coupling. The flies fed on MAG-EPA also had higher electron transport system capacity and mitochondrial oxidative capacities. Moreover, both n-3 PUFAs delayed the occurrence of lipid peroxidation but only flies fed the MAG-EPA diet showed maintenance of superoxide dismutase activity during aging. Our study therefore highlights the potential of n-3 PUFA monoacylglycerides as nutraceutical compounds to delay the onset of senescence by acting directly or indirectly on the mitochondrial metabolism and suggests that Drosophila could be a relevant model for the study of the fundamental mechanisms linking the effects of n-3 PUFAs to aging.

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.