Emilio Fernández

Induction of Glucose‐6‐Phosphate Dehydrogenase by Lipopolysaccharide Contributes to Preventing Nitric Oxide‐Mediated Glutathione Depletion in Cultured Rat Astrocytes

Abstract: Treatment of cultured rat astrocytes with lipopolysaccharide (LPS; 1 μg/ml) increased mRNA expression of glucose‐6‐phosphate dehydrogenase (G6PD), the rate‐limiting step in the pentose phosphate pathway (PPP), in a time‐dependent fashion (0‐24 h). This effect was accompanied by an increase in G6PD activity (1.74‐fold) and in the rate of glucose oxidation through the PPP (6.32‐fold). Inhibition of inducible nitric oxide synthase (iNOS) activity by 2‐amino‐5,6‐dihydro‐6‐methyl‐4H‐1,3‐thiazine (AMT; 50 μM) did not alter the LPS‐mediated enhancement of G6PD mRNA expression or PPP activity. Blockade of nuclear factor κB (NF‐κB) activation by N‐benzyloxycarbonyl‐Ile‐Glu‐(O‐tert‐butyl)‐Ala‐leucinal (1 μM) prevented the expression of both iNOS mRNA and G6PD mRNA, suggesting that iNOS and G6PD are co‐induced by LPS through a common transcriptional pathway involving NF‐κB activation. Incubation of cells with LPS for 24 h increased intracellular NADPH concentrations (1.63‐fold) as compared with untreated cells, but GSH concentrations were not modified by LPS treatment up to 60 h of incubation. However, inhibition of G6PD activity by dehydroepiandrosterone (DHEA; 100 μM), which prevented LPS‐mediated enhancements in PPP activity and NADPH concentrations, caused a 50% decrease in the GSH/GSSG ratio after 24‐36 h and in GSH concentrations after 60 h of incubation. Furthermore, the changes in glutathione concentrations caused by DHEA were abolished by AMT, suggesting that nitric oxide and/or its reactive derivatives would be involved in this process. From these results, we conclude that LPS‐mediated G6PD expression prevents GSH depletion due to nitric oxide and suggest that this phenomenon may be a contributing factor in the defense mechanisms that protect astrocytes against nitric oxide‐mediated cell injury.

CYCLOSPORINE A HEPATOTOXICITY: EFFECT OF PROLONGED TREATMENT WITH CYCLOSPORINE ON BILIARY LIPID SECRETION IN THE RAT

1. The effects of cyclosporine A (CyA) treatment on liver morphology, bile flow and biliary secretion of bile acid, cholesterol and phospholipid and some plasma biochemical indicators of liver function were examined.

Inhibition of biliary glutathione secretion by cyclosporine A in the rat: possible mechanisms and role in the cholestasis induced by the drug.

BACKGROUND/AIMS Biliary glutathione appears to be a major osmotic factor in the generation of bile acid-independent bile flow. This study was designed to investigate its importance in cyclosporine A-induced cholestasis in both acute and short-term-treated rats. METHODS Adult male Wistar rats were treated as follows: (i) with a single i.v. dose of cyclosporine or its vehicle (acute assays); (ii) with cyclosporine, its vehicle or physiological saline, i.p., for 7 days once per day (short-term treatment assays). Bile flow and biliary glutathione levels were determined under anesthesia both before and after intrabiliary hydrolysis of the tripeptide had been inhibited. RESULTS Acute cyclosporine administration, at a dose of 20 mg/kg, brought about an abrupt and marked fall in bile flow and bile acid secretion simultaneously with a rapid decrease in the biliary concentration and secretion rates of total, reduced and oxidized glutathione. When the rats were treated with cyclosporine A for 1 week, at a dose of 10 mg/kg per day, similar cholestatic and inhibitory effects on the biliary secretion of glutathione were noted both before and after the intrabiliary catabolism of the tripeptide had been inhibited with acivicin; in addition, the hepatic content of glutathione was also reduced. The cholestatic effect of the drug was associated with reductions in the four bile flow fractions evaluated: bile acid- and glutathione-dependent bile flow and bile acid- and glutathione-independent bile flow. CONCLUSIONS These findings indicate that cyclosporine-induced cholestasis in the rat is due not only to alterations in the hepatobiliary transport of bile acids but also to an impairment of bile formation dependent on the biliary secretion of glutathione, possibly through inhibition of the canalicular transport of the tripeptide.

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

Summary The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation.

Inhibition of mitochondrial respiration by nitric oxide: Its role in glucose metabolism and neuroprotection

There is an increasing body of evidence demonstrating that inhibition of cytochrome c oxidase by nitric oxide (NO) may be one more step in a signaling cascade involved in the physiologic regulation of cell functions. For example, in both astrocytes and neurons the inhibition of mitochondrial respiration by endogenously produced NO induces transient and modest decreases in cellular ATP concentrations. This mitochondrial impairment may serve as a cellular sensor of energy charges, hence modulating metabolic pathways, such as glycolysis, through AMP‐activated protein kinase (AMPK) in astrocytes. In neurons, the NO derivative peroxynitrite anion triggers signaling pathways leading to glucose oxidation through the pentose‐phosphate pathway to form reducing equivalents in the form of NADPH. The modulation of these metabolic pathways by nitric oxide or its derivatives may be important for understanding the mechanisms by which this free radical affects neuronal death or survival.

DJ1 represses glycolysis and cell proliferation by transcriptionally up-regulating pink1

DnaJ-1 or hsp40/hdj-1 (DJ1) is a multi-functional protein whose mutations cause autosomal recessive early-onset Parkinsons disease (PD). DJ1 loss of function disrupts mitochondrial function, but the signalling pathway, whereby it interferes with energy metabolism, is unknown. In the present study, we found that mouse embryonic fibroblasts (MEFs) obtained from DJ1-null (dj1-/-) mice showed higher glycolytic rate than those from wild-type (WT) DJ1 (dj1+/+). This effect could be counteracted by the expression of the full-length cDNA encoding the WT DJ1, but not its DJ1-L166P mutant form associated with PD. Loss of DJ1 increased hypoxia-inducible factor-1α (Hif1α) protein abundance and cell proliferation. To understand the molecular mechanism responsible for these effects, we focused on phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-induced protein kinase-1 (Pink1), a PD-associated protein whose loss was recently reported to up-regulate glucose metabolism and to sustain cell proliferation [Requejo-Aguilar et al. (2014) Nat. Commun. 5, 4514]. Noticeably, we found that the alterations in glycolysis, Hif1α and proliferation of DJ1-deficient cells were abrogated by the expression of Pink1. Moreover, we found that loss of DJ1 decreased pink1 mRNA and Pink1 protein levels and that DJ1, by binding with Foxo3a (forkhead box O3a) transcription factor, directly interacted with the pink1 promoter stimulating its transcriptional activity. These results indicate that DJ1 regulates cell metabolism and proliferation through Pink1.

The oxidized form of vitamin C, dehydroascorbic acid, regulates neuronal energy metabolism

Vitamin C is an essential factor for neuronal function and survival, existing in two redox states, ascorbic acid (AA), and its oxidized form, dehydroascorbic acid (DHA). Here, we show uptake of both AA and DHA by primary cultures of rat brain cortical neurons. Moreover, we show that most intracellular AA was rapidly oxidized to DHA. Intracellular DHA induced a rapid and dramatic decrease in reduced glutathione that was immediately followed by a spontaneous recovery. This transient decrease in glutathione oxidation was preceded by an increase in the rate of glucose oxidation through the pentose phosphate pathway (PPP), and a concomitant decrease in glucose oxidation through glycolysis. DHA stimulated the activity of glucose‐6‐phosphate dehydrogenase, the rate‐limiting enzyme of the PPP. Furthermore, we found that DHA stimulated the rate of lactate uptake by neurons in a time‐ and dose‐dependent manner. Thus, DHA is a novel modulator of neuronal energy metabolism by facilitating the utilization of glucose through the PPP for antioxidant purposes.

Underestimation of the pentose-phosphate pathway in intact primary neurons as revealed by metabolic flux analysis

The rates of glucose oxidized at glycolysis and pentose–phosphate pathway (PPP) in neurons are controversial. Using [3-3H]-, [1-14C]-, and [6-14C]glucose to estimate fluxes through these pathways in resting, intact rat cortical primary neurons, we found that the rate of glucose oxidized through PPP was, apparently, ∼14% of total glucose metabolized. However, inhibition of PPP rate-limiting step, glucose-6-phosphate (G6P) dehydrogenase, increased approximately twofold the glycolytic rate; and, knockdown of phosphoglucose isomerase increased ∼1.8-fold the PPP rate. Thus, in neurons, a considerable fraction of fructose-6-phosphate returning from the PPP contributes to the G6P pool that re-enters PPP, largely underestimating its flux.

Fuel Supply to the Brain During the Early Postnatal Period

Perinatal time spans over three periods so called the gestational, suckling and weaning period. Gestation ends in the labor which interrupts maternal supply of metabolic substrates giving way to an autonomous metabolism. The fuel supply is rapidly regained by the milk nutrients which supply the newborn with the energy and carbon skeletons required for its development. However, immediately after birth and before the onset of suckling takes place there is a time lapse during in which the newborn has to withstand a unique starvation. This period, herewith referred to the presucking period, is a consequence of incompatibility of the first strokes of ventilation and suckling itself, although more probably it is due to the time elapsed to the compulsory change in the metabolic substrates from the womb to the mammary glands. Nevertheless, during the presuckling period, the newborn has to survive from its own reserves during an unique period of stress and vulnerability. To get through this short but difficult period, the fetus accumulates important energy reserves and adapts its metabolic machinery to the expected changes in its surroundings; the period devoted to this preparation can be called “prepartum” (Figure 1).

Cyclosporin A-Induced Cholestasis in the Rat

SummaryCyclosporin A, a powerful immunosuppressor drug, induces nephrotoxicity and hepatotoxicity. The purpose of this study was to evaluate the ability of S-adenosyl-L-methionine (SAMe) to antagonise the cyclosporin A-induced hepatotoxicity in rats treated with cyclosporin A plus SAMe. Cyclosporin A treatment for 1 or 2 weeks increases plasma bilirubin, alters some plasma biochemical indicators of hepatic and renal function, causes cholestasis and reduces the biliary concentration and secretion of bile acid and other bile components. SAMe pretreatment and simultaneous treatment with SAMe plus cyclosporin A suppresses bilirubin increases in plasma, attenuates cholestasis and totally antagonises the adverse effects of cyclosporin A on bile acid secretion. Although cyclosporin A-induced hepatotoxicity in the rat is a multifactorial phenomenon, we suggest that the hepatoprotective effects of SAMe against cyclosporin A could be related to its regulatory function of membrane lipid composition and fluidity, either alone or combined with stimulation of the hepatic synthesis of thiol compounds.