The present mini-review investigates a new metabolic role for a microsomal membrane bound enzyme, hexose 6 phosphate dehydrogenase (H6PD), which may be involved in chemical energy production, particularly in the nervous system. H6PD represents the counterpart of glucose 6-phosphate dehydrogenase (G6PD), leading to an endoluminal pentose phosphate pathway. Currently, H6PD is considered a producer of reduced equivalents, activating the metabolism and the autocrine/ paracrine effects of glucocorticoid hormones. However, recently, we have demonstrate that H6PD is also expressed in myelin sheath where it may provide the reduced equivalent for the myelin extramitochondrial oxidative phosphorylation (OXPHOS), which produces ATP to support the axonal energy demand. Considering that proteome analysis reported an ectopic localization of OXPHOS machinery also in the microsomal fraction, it is hypothesized that the energy metabolism, involving H6PD and electron transport chain, may happen also in the endoplasmic reticulum. The possibility to produce ATP directly in ER could sustain more efficiently the numerous endoluminal activities.
Keywords: Hexose 6-Phosphate Dehydrogenase; Galactose Catabolism; Endoplasmic Reticulum; Oxidative Phosphorylation
H6PD: Hexose 6-Phosphate Dehydrogenase;
G6PD: Glucose 6-Phosphate Dehydrogenase;
OXPHOS: Oxidative Phosphorylation;
ER: Endoplasmic Reticulum;
ePPP: Endoluminal Pentose Phosphate Pathway;
CNS: Central Nervous System;
11β-HSD1: 11β- Hydroxysteroid Dehydrogenase Type 1;
MS: Multiple Sclerosis;
G6P: Glucose 6 Phosphate
Hexose 6-Phosphate Dehydrogenase: the Past and the Present
Hexose 6-phosphate dehydrogenase (H6PD, E.C. 18.104.22.168) is exclusively located in the endoplasmic reticulum (ER) . Its expression was reported in several rat tissues, including liver, adrenals, spleen, kidney, heart, lungs, muscle, testes, ovaries, prostate, uterus, and intestine [2,3]. Subsequently, H6PD activity was studied in many human tissues including liver, placenta, lymphoid, fibroblasts, adipose, white blood cells, kidney, thymus, pancreas, ovary, testis, skeletal muscle, and lung [4–6].
Protein sequence of H6PD reveals its being a bifunctional enzyme, able to conduct the first two steps of the endoluminal pentose phosphate pathway (ePPP) . In fact, in mice, it was demonstrated that H6PD catalyzes both the oxidation of glucose 6-phosphate (G6P) and the consequently hydrolysis of 6-phosphogluconolactone to 6-phosphogluconate [1,6,8,9]. Considering that there is a high sequence similarity between the murine and human H6PD, it is reasonable to believe that also the human enzyme can conduct the same reactions . Oxidation of G6P to 6- phosphogluconate is associated with the reduction of NADP to NADPH, thereby maintaining adequate levels of reductive cofactors in the oxidizing environment of the ER [11,12]. Moreover, H6PD can also utilize NAD+ as substrate , producing both NADH + H+ instead of NADPH.
H6PD is distinct from its cytosolic homolog, glucose-6-phosphate dehydrogenase (G6PD) by several features. Firstly, H6PD is a microsomal and autosomal protein, while G6PD is a cytosolic and sex-linked enzyme . Moreover, even though literature reports that its substrate is G6P, H6PD is able to oxidize other phosphorylated and non-phosphorylated hexoses, faster than glucose under physiological conditions . H6PD displays a low KM for D-galactose, and its affinity is good also for other phosphorylated and non-phosphorylated hexoses: galactose 6- phosphate, 2-deoxyglucose, glucose 6-phosphate, 2-deoxyglucose 6-phosphate, glucosamine 6- phosphate, and glucose 6-sulfate [9,10].
Presently, the main role ascribed to H6PD is the generation of NADPH to sustain the steroid hormone and drug metabolism . In particular, H6PD would be associated with another endoluminal enzyme, 11β-hydroxysteroid dehydrogenase type 1, (11β-HSD1), an enzyme responsible for the activation and inactivation of glucocorticoids . 11β-HSD1 is a bidirectional enzyme highly expressed in liver and adipose tissue, but in intact cells it displays predominantly oxo-reductase activity [15,16]. The reduced equivalents produced by H6PD could cooperate with other intraluminal reductase [17,18], as, for example, the NADPH-cytochrome c reductase to sustain the microsomal electron transport system .
Hexose 6-Phosphate Dehydrogenase and Galactose: a Novel Energetic Role in Myelin Sheath Metabolism
Recently, we have proposed a novel role for H6PD  in the extramitochondrial energy metabolism of myelin sheath [21– 28]. We have demonstrated that H6PD is expressed in myelin and is able to metabolize galactose (Gal) to sustain the ATP synthesis through OXPHOS. In particular, NADPH, produced by H6PD in the presence of Gal, would become a respiring substrate for Complex I , supposing a direct link among H6PD and electron transport chain in myelin sheath. Notably, Gal has been traditionally considered a preferential substrate for the OXPHOS , being commonly used to “bypass glycolysis” and deliver the reducing equivalents directly to the electron transport chain (ETC). Moreover, Gal can increase the OXPHOS proteins expression .
Considering that myelin contains all the enzymes involved in glycolysis and Krebs Cycle , it is possible to hypothesize that the galactonate, produced by H6PD, may be converted in xilulose 5-phopshate, by a dehydrogenase, entering the ePPP or be transformed into pyruvate and glyceraldehyde 3-phosphate, by dehydratase and aldolase activities (see the schematic in Figure 1). The presence of a microsomal enzyme in the sheath is consistent with our hypothesis that the OXPHOS machinery is transferred from mitochondria to myelin through the ER and Golgi apparatus [23,28,31].
Moreover, the expression of H6PD in the Central Nervous System (CNS) could clarify why the Gal metabolism in brain is so consistent , confirming the data about the pivotal role of ePPPn CNS [33,34]. In particular, a recent study has observed that, even though the liver shows the highest initial capacity for the uptake and metabolization of galactose, the brain has the highest ratio of galactose metabolites to galactose, 50% higher than in kidney and 20% in liver are in brain . The same Authors observed that in rats treated with galactose, the concentration of UDP-glucose remained nearly unchanged in brain, while UDP-galactose showed an increment , even though the activity of galactokinase and uridyltransferase was lower with respect to that observed in liver . These data suggest that galactose could be used as a metabolic substrate in brain, using a different pathway with respect to that present in liver. In fact, currently, it is believed that the D-gal can be used as an energy substrate only after its conversion to glucose in the liver , via the Leloir pathway, but this pathway needs first of all the conversion of the beta anomer to an alpha one . By contrast, we retain that Gal may be catabolized by another pathway involved H6PD and the ePPP.
Figure1. Galactose Catabolism by H6PD and Pentose Phosphate Pathway.
The scheme represents a possible Galactose (Gal) catabolism pathway, involving H6PD and Pentose Phosphate Pathway (PPP).
Firstly, Gal would be oxidized by H6PD to galactonate and to xylulose, producing NADPH. These products, going on the PPP, could provide pyruvate and glyceraldehyde 3-phosphate, which could metabolized by other energy metabolic pathway, as Krebs cycle and glycolysis. Enzymes are identified following the Enzyme Classification (EC) numbering.
Concerning the CNS Gal metabolism, it is important to note that galactose passes the ematoencephalyc barrier and enters in the cell by not only GLUT4, as glucose, but also by GLUT3, an hexose transporter no-insulin dependent . Notably, it was found that the expression in mice of GLUT3, increases after 14 days from birth and reaches a peak at 21 days from birth , following the myelinogenesis process. In fact, myelin starts to develop at 5-6 days of age, accelerates around the 14th day after birth, being complete in about 4 weeks , suggesting a link among myelin sheath and Gal utilization. Considering that also in humans myelination begins after the birth , it is possible to presume that a considerable part of galactose, from milk, is not converted in glucose by liver, but is used principally to the myelin sheath formation and energy metabolism.
The H6PD gene was found associated to Multiple Sclerosis (MS) , a demyelinating disease of CNS, confirming that this enzyme plays an important role in myelin sheath and brain metabolism. Moreover, a clinical study on patients affected by MS demonstrated that administration of galactose improved the life quality and performance of patients, reverting the most invalidating symptoms of the demyelinating disease . Authors ascribed the positive effect of galactose exclusively to a fast remyelination, due to the need to produce galactocerebrosides. Conversely, we propose that galactose could also play a metabolic role, enhancing the myelin OXPHOS activity. Beneficial effect of one month oral galactose in a dose ranging from 100 to 300 mg/kg/day was shown also in the streptozotocin- induced (STZ-icv) rat, considered a model of sporadic Alzheimer’s disease .
The Energetic Role of Hexose 6-Phosphate Dehydrogenase Activity in the ER
We believe that the link between H6PD activity and the extramitochondrial OXPHOS in myelin could be extended also to ER of all cell types.
ER is known to modulate the energy metabolism of the cell, integrating various metabolic signals and pathways so to regulate lipid, glucose, cholesterol, and protein metabolism . ER is considered a separate metabolic compartment because it displays substrate specificity and selective transport ability of a series of possible substrates and co-factors from the cytosol . ER is a main site of protein synthesis and post-translational protein modifications and, together with the Golgi apparatus, it takes part to the correct folding, transport and of proteins . All these activities demand a lot of chemical energy, in the form of ATP, which is currently considered to be dispensed by the mitochondrion. Conversely, following our challenging line of thought, according to which H6PD would play a peculiar metabolic role if linked to an extra-mitochondrial OXPHOS, on a membrane, it is possible to hypothesize that the ER could be a site of ectopic aerobic ATP production. In fact, a proteomic analysis of microsomal fraction reported that out of 1035 identified proteins, 304 belonged to mitochondria, with a particular representation of those involved in the OXPHOS activity . This is suggestive of ER containing an extramitochondrial energy production ability, the reduced equivalents necessary being dispensed by H6PD. Normally, mitochondria OXPHOS is feed by NADH and FADH2 produced by Krebs cycle; by contrast, in ER, glycolysis and Krebs cycle are not necessary, because H6PD could provide directly the reduced equivalents to Complex I, making the energy synthesis faster. Moreover, H6PD is able to oxidize several hexoses, other than glucose, allowing a more efficient energy production.
Several researches have demonstrated that the energy metabolism is negatively influenced by the ER stress and the unfolded protein response (UPR) [44,48–50]. For example, Gregor et al described an association among ER and adipocyte dysfunction, leading to develop metabolic disease, as diabetes and obesity . Moreover, the knout of H6PD gene induces a display a progressive skeletal myopathy in mice, with post-mortem muscle tissue weights reduced by 20–40% , with a significant fiber type switch from type II (fast) to type I (slow), suggesting that H6PD is involved not only in the steroid hormone and in drug metabolism.
The wide tissue distribution of H6PD suggests that this membrane- bound enzyme could be considered a pivotal housekeeping enzyme, critically involved in several biochemical pathways, from the maintenance of the redox homeostasis of the ER lumen to the aerobic ATP production.
This new vision about H6PD renders this enzyme a new candidate for future therapies for diseases linked to ER impairment.