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Mercaptosuccinate and diethyl maleate were dissolved in distilled water and prepared daily. At the end of the incubation, 1. The tubes were loosely capped and incubated in a boiling water bath for 45 min. Following cooling, 2 ml of n -butanol was added to each tube to extract the TBA-MDA adduct, and the tubes were vortexed before being left to stand for 10 min to ensure complete butanol separation.
Approximately 1. A separate tube was prepared for each measurement six tubes per experimental condition to avoid the need to open tubes and admit air until the required time had elapsed. We endeavored to analyze at least motile sperm per sample. This was impossible in some samples exposed to high BiCNU concentrations, however, because too few motile sperm were present.
Data were first tested for normality of distribution with the Kolmogorov-Smirnov test and for homogeneity of variance with the Levene test. Normally distributed data with equal variance between groups were analyzed by one- or two-way ANOVA as appropriate.
The Scheffe test was used for post-hoc testing where it was appropriate to analyze differences between all groups, and the Dunnett test was used when comparisons were made with a control. Nonnormally distributed data were tested with the nonparametric Kruskal-Wallis test. Cumene hydroperoxide is an excellent substrate for glutathione peroxidase and a weak oxidizing agent.
We predicted that low to moderate concentrations would increase flux through glutathione peroxidase, with a consequent increase in flux through the pentose phosphate pathway. Toxic effects, anticipated at high concentrations, would be indicated by a decrease in the rate of glycolysis.
These results were not affected by any low-level leukocyte contamination in the sperm suspensions, because for experiments in which sperm suspensions were divided in two and one part was denuded of leukocytes by treatment with anti-CDcoated Dynabeads, the results from the intact and the leukocyte-depleted suspensions were identical data not shown.
Effect of increasing concentrations of cumene hydroperoxide on the rate of metabolism through a the pentose phosphate and b the glycolytic pathways in human sperm.
The rates of pentose phosphate and glycolytic activity were assessed by the release of 14 CO 2 and 3 H 2 O, respectively.
Effect of increasing concentrations of H 2 O 2 on the pentose phosphate pathway and glycolytic flux in human sperm. In a similar way, pentose phosphate pathway activity was increased by the addition of 1 mM xanthine and increasing amounts of XO Fig. This resulted from the effects of H 2 O 2 , because the effect was blocked by catalase Fig. Xanthine increased pentose phosphate pathway activity above the basal rate even in the absence of added XO, implying that sperm have endogenous XO activity Fig.
This increase was also attributed to H 2 O 2 , because it was also blocked by catalase Fig. Stimulation of a the pentose phosphate pathway flux in human sperm by the superoxide-generating system, xanthine plus xanthine oxidase, and the effects of b catalase and c SOD. Flux through the pentose phosphate pathway was measured as described for Figure 1. Ascorbate alone increased pentose phosphate pathway flux from 0. The addition of FeSO 4 caused only a small and statistically insignificant increase above that produced by ascorbate alone, to 0.
The effect of ascorbate did not depend on the presence of endogenous iron, because it was unaffected by the addition of either 0. Mercaptosuccinate is an inhibitor of glutathione peroxidase that has been used previously to inhibit this enzyme in sperm.
Inhibition of basal and cumene hydroperoxide-stimulated pentose phosphate pathway flux in human sperm by BiCNU. The pentose phosphate pathway flux was measured during the subsequent hour by the release of 14 CO 2 as described for Figure 1.
Influence of oxidative stress on lipid peroxidation in human sperm. Increased lipid peroxidation in human sperm incubated with increasing concentrations of BiCNU. The effect of oxygen could be seen more clearly by examining the ratio between motility under oxygen and motility under nitrogen. Separate tubes were prepared for each time point. These observations clearly demonstrate that flux through the pentose phosphate pathway in human sperm increases in response to moderate oxidative stress.
Such levels of stress have no effect on glycolytic flux, supporting the view that the stimulation is specific and that antioxidant defenses in human sperm are capable of protecting sensitive sulfhydryl enzymes, notably glyceraldehyde 3-phosphate dehydrogenase, against oxidative stress [ 26 ]. Were the pentose phosphate pathway responding to a stimulatory effect of moderate oxidative stress, we would expect a parallel increase in glycolysis to meet the increased energy demand [ 23 ].
The capacity of the antioxidant pathway is limited, and defenses are overwhelmed by high levels of oxidative stress that decrease flux through both the pentose phosphate pathway and glycolysis and through reduced sperm motility. Evidence from other systems suggests that the addition of cumene hydroperoxide is an effective way to increase flux through glutathione peroxidase and to study the subsequent response of the pentose phosphate pathway [ 27 , 28 ].
The glutathione peroxidase-glutathione reductase-pentose phosphate pathway axis is important in limiting the oxidative damage suffered by human sperm, because blocking glutathione reductase with BiCNU made the pentose phosphate pathway unable to respond to increased demand from glutathione peroxidase and made the cells more vulnerable to lipid peroxidation and loss of motility under aerobic conditions.
Our data do not allow the capacity of the pathway to be estimated, because it was necessary to use only 0. Estimates of the maximum activity of glucose 6-phosphate dehydrogenase activity in human sperm range from 3. A major anomaly in our results is the failure of mercaptosuccinate to block the increase in pentose phosphate pathway flux induced by cumene hydroperoxide. Mercaptosuccinate has been shown to inhibit glutathione peroxidase activity in human sperm with a concomitant increase in sensitivity to lipid peroxidation [ 14 , 18 , 31 ].
It is possible that an alternative route of glutathione or NADPH oxidation is linked to the presence of cumene hydroperoxide. It is unlikely that this would be glutathione peroxidase 5 derived from the epididymis, however, because this enzyme is present only as an inactive splice variant in humans [ 32 ]. Moreover, some nonselenium glutathione peroxidases are sensitive to mercaptosuccinate [ 33 ]. Another possibility is that the residual glutathione peroxidase activity is sufficient to account for the rather modest rate of pentose phosphate pathway activity that was achieved in the presence of a low glucose concentration.
In our hands, however, mercaptosuccinate had no effect on sperm viability or motility [ 34 ]. All our other observations are consistent with the metabolism of peroxides by glutathione peroxidase leading to the formation of oxidized glutathione that is regenerated by glutathione reductase leading to a demand for NADPH production through the pentose phosphate pathway.
An important concern in all experiments about ROS metabolism by human sperm is to what extent the results reflect the presence of contaminating leukocytes. Here, removal of leukocytes with anti-CDcoated Dynabeads had no effect on pentose phosphate pathway activity, so we can be confident that the data represent the metabolic capacity of sperm cells. With this in mind, it is interesting that addition of catalase decreased the basal flux through the pentose phosphate pathway, implying that even in sperm suspensions with a very low level of leukocyte contamination, the sperm need to detoxify hydrogen peroxide.
Recent reports suggest that human sperm do not contain measurable NADPH oxidase activity [ 30 , 35 ], although multiple plasma membrane redox systems are present [ 36 ]. The hydrogen peroxide likely is derived from the mitochondria, from peroxidase enzymes, or from one or more of the plasma membrane redox systems.
By contrast, although diethyl maleate, which is presumed to complex cell glutathione, inhibited the increase in pentose phosphate pathway flux produced by cumene hydroperoxide, it had no effect on the basal rate, suggesting that the latter rate is independent of glutathione and, by inference, glutathione peroxidase.
The basal rate varied between sperm from different donors and between different ejaculates from the same donor. This probably reflects differences in oxidative stress and activity of other redox systems [ 36 ], but it will also be influenced by other demands for NADPH, the existence of which is suggested by residual pentose phosphate pathway activity in the presence of high concentrations of BiCNU Fig. We were anxious to establish if the pathway would respond to lipid peroxidation, and we tested the effect of exposing the sperm to ascorbate plus ferrous iron.
Ascorbate increased pentose phosphate pathway flux. However, no significant additional effect of iron was found even though iron greatly increased malondialdehyde release when added to the HBSS in the subsequent lipid peroxidation assay [ 34 ].
These observations are consistent with ascorbate acting as a pro-oxidant and promoting lipid peroxidation de novo, whereas the ferrous iron promoter initiates free radical formation from stabilized lipid peroxides in the cell membrane, leading to malondialdehyde production proportionate to preexisting but stabilized membrane peroxidation [ 24 ].
On this premise, the pentose phosphate pathway in human sperm is able to respond to lipid peroxidation as well as to H 2 O 2 and synthetic organic peroxides. The ability of H 2 O 2 to induce lipid peroxidation as well as acting as a glutathione peroxidase substrate may explain why it can increase pentose phosphate pathway flux to a greater extent than cumene hydroperoxide can.
A possible explanation is that partial inhibition of glutathione reductase leads to an increase in the proportion of oxidized glutathione. This might stimulate the activity of glucose 6-phosphate dehydrogenase, whereas the increased substrate concentration maintains flux through glutathione reductase. Alternatively, it is conceivable that low concentrations of BiCNU might stimulate one of the plasma membrane redox systems [ 36 ].
This was largely reflected in its effect on lipid peroxidation Fig. The ratios between motility under oxygen and under nitrogen Fig. However, BiCNU had a significant inhibitory effect on motility even under nitrogen. To conclude, we believe our data demonstrate that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system in human sperm can respond dynamically to oxidative stress and is capable of protecting the cells against oxidative damage.
We are grateful for help and support from the late Prof. Macleod J. Figure 3. The role of the PPP in insulin secretion. Diabetes can lead to diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, diabetic macroangiopathy and other chronic complications.
Oxidative stress can cause these complications by activating the hexosamine pathway, the advanced glycation end products AGEs pathway and the diacylglycerol DAG -protein kinase C PKC pathway Hyperglycemia decreases G6PD activity through the activation of protein kinase A PKA and increase of intracellular oxidative stress, leading to chronic kidney injury, and diabetic kidney disease DKD 43 — Moreover, overexpression of G6PD in endothelial cells prevents diabetic cardiomyopathy by decreasing ROS accumulation and increasing endothelial cell viability TKT plays an important role in preventing hyperglycemia-induced vascular cell dysfunction Thiamine deficiency and decreased TKT activity has been reported to contribute to diabetic complications Low plasma thiamine was found in patients with DKD and diabetic rats.
After high-dose thiamine therapy, the progression of proteinuria and microalbuminuria was reversed in both patients and animal models, indicating that regulating the activity of TKT may be a promising therapy in treating DKD 47 , 49 , Benfotiamine, a lipid-soluble thiamine derivative, can prevent diabetic retinopathy and cardiomyopathy as well as accelerate the healing of diabetic limbs by activating TKT 51 — However, benfotiamine did not show promising efficacy in phase II and IV trials for the treatment of DKD or diabetic peripheral nerve function 54 , Therefore, other transketolase activators await further investigation.
Various enzymes in the PPP have been shown to be potential targets in cancer therapy. These proteins not only function as metabolic enzymes, but also participate in the regulation of other cellular activities. Therefore, we will summarize recent findings in upstream signaling pathways regulating PPP enzymes in cancer initiation and progression Table 1.
Up-regulation of the G6PD level or activity is often observed in many kinds of cancer 79 — Several signaling pathways have been identified to be responsible for promoting G6PD expression or activity in cancer cells Figure 4. TAp73, a member of the p53 family which is often overexpressed in cancers, supports tumor growth by inducing G6PD expression 56 , Nuclear factor E2-related factor 2 NRF2 is a transcription factor regulated by oxidative stress.
In addition, some post-translational modifications such as phosphorylation, acetylation, O-GlcNAcylation and ubiquitination affect the activity of G6PD 59 , 67 , 69 , Figure 4.
Regulation of G6PD in cancers. Several signaling pathways have been identified to be responsible for promoting G6PD expression or activity in cancer cells. These signaling pathways interact with each other, adding complexity to the regulation of G6PD. The level of G6PD often negatively correlates to the prognosis of cancer patients Suppression of G6PD induces cellular senescence in hepatocellular carcinoma HCC cells and leads to intracellular oxidative stress, making cancer cells sensitive to chemotherapy 60 , Interestingly, elevated G6PD is not observed in liver cirrhosis which is a main cause of liver cancer, indicating that G6PD might play an important role in promoting malignant transformation However, the role of 6PGL in cancer remains to be elucidated.
In addition to its function as a metabolic enzyme, 6PGD also regulates cell metastasis by promoting phosphorylation of c-Met Post-translational modification of 6PGD is important for cancer cell proliferation and tumor growth.
Acetylation of 6PGD plays a key role in coordinating redox homeostasis, lipogenesis and glycolysis Patients with lower levels of 6PGD Y phosphorylation have longer median survival time Aberrant expression of 6PGD can accelerate cancer cell proliferation and induce resistance to chemical or radical therapy 72 , 94 — All these findings suggest that inhibiting the expression or activity of 6PGD might be a promising therapeutic strategy for cancer.
Kras G12D regulates the non-oxidative but not oxidative PPP to provide cancer cells with sufficient R5P for nucleotide biosynthesis RPI promotes tumor growth and colony formation by negatively modulating protein phosphatase 2A PP2A to activate extracellular signal-regulated kinase ERK signaling pathways In zebrafish, overexpression of RPI contributes to fatty liver, liver cirrhosis and cell proliferation High level of RPI is reported to predict negative clinical outcomes of colorectal cancer patients.
The expression of TKT is elevated in many types of cancer 75 — 78 , 83 , Patients with pancreatic cancer have higher levels of serum fructose which induces TKT expression to drive nucleic acid synthesis in cancer cells Despite the accumulation of R5P, knockdown of TKT suppresses tumor growth and sensitizes cancer cells to chemotherapy Recent work suggests that TKT promotes genome instability by regulating nucleotide biosynthesis during liver injury and cancer initiation In addition, TKT can regulate cell cycle and promote the viability and proliferation of cancer cells independent of its enzyme activity.
Moreover, higher TALDO expression often indicates poorer clinical outcomes and more resistance to trastuzumab therapy in breast cancer. When human epidermal growth factor receptor 2 HER2 signaling is inhibited, breast cancer cells rely on the non-oxidative arm of the PPP to replenish the oxidative arm.
NADPH, a key intracellular reductant, is required for glutathione system and other ROS scavengers to maintain the redox homeostasis Therefore, the PPP serves as an ideal target for regulating the redox homeostasis in metabolic diseases and cancer. The PPP regulates insulin secretion. Insulin promoting the growth and proliferation of cells is one of the mechanisms underlying increased cancer risk in obese and diabetic patients , Chronic inflammation is a well-known hallmark of cancer and insulin resistance.
Obesity-related inflammation is believed to create a microenvironment contributing to the initiation and progression of cancer In turn, cancer cells secrete cytokines to recruit macrophages, leading to cancer-related inflammation, which plays an important role in cancer cell migration and invasion , The PPP plays a critical role in type 2 diabetes and cancer.
XT designed and revised the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor is currently co-organizing a Research Topic with one of the authors XT, and confirms the absence of any other collaboration. We would like to thank all members in the Tong laboratory for their helpful suggestions.
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