Glycerol Phosphate Shuttle Research Articles

Immp2l Enhances the Structure and Function of Mitochondrial Gpd2 Dehydrogenase.

'Inner mitochondrial membrane peptidase 2 like' (IMMP2L) is a nuclear-encoded mitochondrial peptidase that has been conserved through evolutionary history, as has its target enzyme, 'mitochondrial glycerol phosphate dehydrogenase 2' (GPD2). IMMP2L is known to cleave the mitochondrial transit peptide from GPD2 and another nuclear-encoded mitochondrial respiratory-related protein, cytochrome C1 (CYC1). However, it is not known whether IMMP2L peptidase activates or alters the activity or respiratory-related functions of GPD2 or CYC1. Previous investigations found compelling evidence of behavioural change in the Immp2lKD-/- KO mouse, and in this study, EchoMRI analysis found that the organs of the Immp2lKD-/- KO mouse were smaller and that the KO mouse had significantly less lean mass and overall body weight compared with wildtype littermates (p < 0.05). Moreover, all organs analysed from the Immp2lKD-/- KO had lower relative levels of mitochondrial reactive oxygen species (mitoROS). The kidneys of the Immp2lKD-/- KO mouse displayed the greatest decrease in mitoROS levels that were over 50% less compared with wildtype litter mates. Mitochondrial respiration was also lowest in the kidney of the Immp2lKD-/- KO mouse compared with other tissues when using succinate as the respiratory substrate, whereas respiration was similar to the wildtype when glutamate was used as the substrate. When glycerol-3-phosphate (G3P) was used as the substrate for Gpd2, we observed ~20% and ~7% respective decreases in respiration in female and male Immp2lKD-/- KO mice over time. Together, these findings indicate that the respiratory-related functions of mGpd2 and Cyc1 have been compromised to different degrees in different tissues and genders of the Immp2lKD-/- KO mouse. Structural analyses using AlphaFold2-Multimer further predicted that the interaction between Cyc1 and mitochondrial-encoded cytochrome b (Cyb) in Complex III had been altered, as had the homodimeric structure of the mGpd2 enzyme within the inner mitochondrial membrane of the Immp2lKD-/- KO mouse. mGpd2 functions as an integral component of the glycerol phosphate shuttle (GPS), which positively regulates both mitochondrial respiration and glycolysis. Interestingly, we found that nonmitochondrial respiration (NMR) was also dramatically lowered in the Immp2lKD-/- KO mouse. Primary mouse embryonic fibroblast (MEF) cell lines derived from the Immp2lKD-/- KO mouse displayed a ~27% decrease in total respiration, comprising a ~50% decrease in NMR and a ~12% decrease in total mitochondrial respiration, where the latter was consistent with the cumulative decreases in substrate-specific mediated mitochondrial respiration reported here. This study is the first to report the role of Immp2l in enhancing Gpd2 structure and function, mitochondrial respiration, nonmitochondrial respiration, organ size and homeostasis.

Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis

The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.

Glycerol-3-Phosphate Shuttle Is a Backup System Securing Metabolic Flexibility in Neurons.

Electrical activity in neurons is highly energy demanding and accompanied by rises in cytosolic Ca2+ Cytosolic Ca2+, in turn, secures energy supply by pushing mitochondrial metabolism either through augmented NADH (nicotinamide adenine dinucleotide) transfer into mitochondria via the malate-aspartate shuttle (MAS) or via direct activation of dehydrogenases of the TCA cycle after passing into the matrix through the mitochondrial Ca2+ uniporter (MCU). Another Ca2+-sensitive booster of mitochondrial ATP synthesis is the glycerol-3-phosphate shuttle (G3PS), whose role in neuronal energy supply has remained elusive. Essential components of G3PS are expressed in hippocampal neurons. Single neuron metabolic measurements in primary hippocampal cultures derived from rat pups of either sex reveal only moderate, if any, constitutive activity of G3PS. However, during electrical activity neurons fully rely on G3PS when MAS and MCU are unavailable. Under these conditions, G3PS is required for appropriate action potential firing. Accordingly, G3PS safeguards metabolic flexibility of neurons to cope with energy demands of electrical signaling.SIGNIFICANCE STATEMENT Ca2+ ions are known to provide a link between the energy-demanding electrical activity and an adequate ATP supply in neurons. To do so, Ca2+ acts both from outside and inside of the mitochondrial inner membrane. Neuronal function critically depends on this regulation, and its defects are often found in various neurologic disorders. Although interest in neuronal metabolism has increased, many aspects thereof have remained unresolved. In particular, a Ca2+-sensitive NADH (nicotinamide adenine dinucleotide) shuttling system, the glycerol-3-phosphate shuttle, has been largely ignored with respect to its function in neurons. Our results demonstrate that this shuttle is functional in hippocampal neurons and safeguards ATP supply and appropriate action potential firing when malate aspartate shuttle and mitochondrial Ca2+ uniporter are unavailable, thereby ensuring neuronal metabolic flexibility.

Saturation of the mitochondrial NADH shuttles drives aerobic glycolysis in proliferating cells

Proliferating cells exhibit a metabolic phenotype known as "aerobic glycolysis," which is characterized by an elevated rate of glucose fermentation to lactate irrespective of oxygen availability. Although several theories have been proposed, a rationalization for why proliferating cells seemingly waste glucose carbon by excreting it as lactate remains elusive. Using the NCI-60 cell lines, we determined that lactate excretion is strongly correlated with the activity of mitochondrial NADH shuttles, but not proliferation. Quantifying the fluxes of the malate-aspartate shuttle (MAS), the glycerol 3-phosphate shuttle (G3PS), and lactate dehydrogenase under various conditions demonstrated that proliferating cells primarily transform glucose to lactate when glycolysis outpaces the mitochondrial NADH shuttles. Increasing mitochondrial NADH shuttle fluxes decreased glucose fermentation but did not reduce the proliferation rate. Our results reveal that glucose fermentation, a hallmark of cancer, is a secondary consequence of MAS and G3PS saturation rather than a unique metabolic driver of cellular proliferation.

VMAX OF MITOCHONDRIAL ELECTRON SHUTTLES IN RAT SKELETAL MUSCLE AND LIVER

Aerobic glycolytic flux demands a commensurate net transport of reducing equivalents (e-) into mitochondria (MITO). However, because the MITO matrix redox potential is maintained more negative than the cytosol, net e-shuttling requires the application of an energetic driving force. Two well known e- shuttles, the malate-aspartate (MA) and glycerol-3-phosphate (G3P) shuttles, are driven either by delta psi (MA) or via linkage to a more positive FAD-linked redox center (G3P). Recently lactate has been proposed as an additional molecular carrier of e- into MITO. We evaluated the catalytic potentials of these three shuttling systems in MITO isolated from soleus (type I) and superficial vastus (type IIb) rat skeletal muscle and also liver (n = 5 MITO preps per tissue). MITO were incubated in KCl-based media at 37C while O2 consumption (Jo) was measured polarographically. In the presence of saturating ADP, MA shuttle Vmax was assessed using 10 mM glutamate + 1 mM malate + 1 mM arsenite, the G3P shuttle using 10 mM G3P, and the lactate shuttle as 10 mM lactate + 1 mM malate. Pyruvate 1 mM + malate 1 mM (P+M) was also assessed. Data are presented in units of nmol O2/min/mg mito protein as means + SE, and are listed in the following order for each tissue: P+M; MA Shuttle; G3P Shuttle; Lactate Shuttle. Type I: 712+8; 411+51; 11.0+1.3; 9.6+5.2. Type IIb: 639+69; 276+30; 259+9; 4.2+1.8. Liver: 35.2+4.5; 37.1+7.8; 2.5+0.6; 4.0+1.4. Type IIb MITO demonstrated high Jo for both the MA and G3P shuttles, and these rates were fully additive when both shuttles were active (not shown). Lactate gave Jo of 1.5% and 1.6% the MA and G3P shuttles, respectively. Type I and liver MITO had low Jo with G3P, but high MA shuttle Vmax, as previously shown. Laclate Jo was 2.3% and 10.8% of MA shuttle Jo in type I and liver, respectively. We conclude that: 1) the results are consistent with energetically-driven redox separation of the cytosol from the matrix and 2) MITO in fast glycolytic muscle are robustly equipped to shuttle e- from the cytosol. Supported by NSF #IBN-0116997.