Glucose Transport and Transporters in the Endomembranes.

Glucose is a basic nutrient in most of the creatures; its transport through biological membranes is an absolute requirement of life. This role is fulfilled by glucose transporters, mediating the transport of glucose by facilitated diffusion or by secondary active transport. GLUT (glucose transporter) or SLC2A (Solute carrier 2A) families represent the main glucose transporters in mammalian cells, originally described as plasma membrane transporters. Glucose transport through intracellular membranes has not been elucidated yet; however, glucose is formed in the lumen of various organelles. The glucose-6-phosphatase system catalyzing the last common step of gluconeogenesis and glycogenolysis generates glucose within the lumen of the endoplasmic reticulum. Posttranslational processing of the oligosaccharide moiety of glycoproteins also results in intraluminal glucose formation in the endoplasmic reticulum (ER) and Golgi. Autophagic degradation of polysaccharides, glycoproteins, and glycolipids leads to glucose accumulation in lysosomes. Despite the obvious necessity, the mechanism of glucose transport and the molecular nature of mediating proteins in the endomembranes have been hardly elucidated for the last few years. However, recent studies revealed the intracellular localization and functional features of some glucose transporters; the aim of the present paper was to summarize the collected knowledge.

Species-Specific Glucose-6-Phosphatase Activity in the Small Intestine-Studies in Three Different Mammalian Models.

Besides the liver, which has always been considered the major source of endogenous glucose production in all post-absorptive situations, kidneys and intestines can also produce glucose in blood, particularly during fasting and under protein feeding. However, observations gained in different experimental animals have given ambiguous results concerning the presence of the glucose-6-phosphatase system in the small intestine. The aim of this study was to better define the species-related differences of this putative gluconeogenic organ in glucose homeostasis. The components of the glucose-6-phosphatase system (i.e., glucose-6-phosphate transporter and glucose-6-phosphatase itself) were analyzed in homogenates or microsomal fractions prepared from the small intestine mucosae and liver of rats, guinea pigs, and humans. Protein and mRNA levels, as well as glucose-6-phosphatase activities, were detected. The results showed that the glucose-6-phosphatase system is poorly represented in the small intestine of rats; on the other hand, significant expressions of glucose-6-phosphate transporter and of the glucose-6-phosphatase were found in the small intestine of guinea pigs and homo sapiens. The activity of the recently described fructose-6-phosphate transporter-intraluminal hexose isomerase pathway was also present in intestinal microsomes from these two species. The results demonstrate that the gluconeogenic role of the small intestine is highly species-specific and presumably dependent on feeding behavior (e.g., fructose consumption) and the actual state of metabolism.

Early rehabilitation for severe acquired brain injury in intensive care unit: multicenter observational study.

BACKGROUND: The increased survival after a severe acquired brain injury (sABI) raise the problem of making most effective the treatments in Intensive Care Unit (ICU)/Neurointensive Care Unit (NICU), also integrating rehabilitation care. Despite previous studies reported that early mobilization in ICU was effective in preventing complications and reducing hospital stay, few studies addressed the rehabilitative management of sABI patients in ICU/NICU. AIM: To collect clinical and functional data about the early rehabilitative management of sABI patients during ICU/NICU stay. DESIGN: Prospective, observational, multicenter study. SETTING: Fourteen facilities supplied by intensive neurorehabilitation units and ICU/NICUs. POPULATION: Consecutive sABI patients admitted to ICU/NICU. METHODS: Patients were evaluated at admission and then every 3-5 days. Clinical, functional and rehabilitative data, including Glasgow Coma Scale (GCS), Disability Rating Scale (DRS), The Rancho Los Amigos Levels of Cognitive Functioning Scale (LCF), Early Rehabilitation Barthel Index (ERBI), Glasgow Outcome scale (GOS) and Functional Independence Measure (FIM) were collected. RESULTS: One hundred and two patients (F/M 44/58) were enrolled. The mean duration of ICU stay was 24.7±13.9 days and the first rehabilitative evaluation occurred after 8.7±8.8 days. Regular postural changes and multijoint mobilization were prescribed in 63.7% and 64.7% cases, respectively. The mean session duration was 38±11.5 minutes. Swallowing evaluation was performed in 14.7% patients, psychological support was provided to 12.7% of patients' caregivers, while 17.6% received a psycho-educational intervention, and 28.4% were involved in interdisciplinary team meetings. The main discharge destinations were Severe Acquired Brain Injury rehabilitation units for 43.7%, intensive neurorehabilitation units for 20.7%. CONCLUSIONS: Data showed that early rehabilitation was not diffusely performed in sABI subjects in ICU/NICU and rehabilitative interventions were variable; one-third of subjects were not referred to dedicated rehabilitation unit at discharge. CLINICAL REHABILITATION IMPACT: The study stresses the need to spread and implement a rehabilitative culture also for critical ill patients due to neurological diseases.

Vitamin E Improves Clinical Outcome of Patients Affected by Glycogen Storage Disease Type Ib.

BACKGROUND: It has been suggested, on a few GSD1b patients, that vitamin E improves neutrophil count and reduces frequency and severity of infections.The main objective of the present study was to investigate the efficacy of vitamin E on the neutropenia, neutrophil dysfunction and IBD in the entire Italian caseload of GSD1b patients. PATIENTS AND METHODS: Eighteen GSD1b patients, median age at the time of the study protocol 14.5 (range, 0.6-42 years), were enrolled from four Italian referral centres for metabolic diseases. For the evaluation of the efficacy of vitamin E, neutrophil count and function, frequency of infections needing hospitalization and inflammatory bowel activity were evaluated periodically all over one year before and during vitamin E therapy. RESULTS: Frequency (1.5 ± 0.1 vs. 6.0 ± 0.6, p = 0.003) and severity of infections (2.2 ± 0.2 vs. 3.7 ± 0.4, p = 0.003) were lower and mean value of neutrophil count (1,583 ± 668 vs. 941 ± 809, p = 0.03) higher during vitamin E supplementation. Neutrophil function results improved during vitamin supplementation. PCDAI showed a significant reduction in the inflammatory activity during vitamin E supplementation (9 ± 1.4 vs. 13 ± 1.2, p = 0.006). In seven patients G-CSF requirement decreased and the dose was reduced after the end of the study.In conclusion, our study demonstrated the efficacy of vitamin E supplementation. Vitamin E has evident advantages as compared to G-CSF, as it can be assumed orally, and it has not been associated with severe side effects.

Subcellular compartmentation of ascorbate and its variation in disease states.

Beyond its general role as antioxidant, specific functions of ascorbate are compartmentalized within the eukaryotic cell. The list of organelle-specific functions of ascorbate has been recently expanded with the epigenetic role exerted as a cofactor for DNA and histone demethylases in the nucleus. Compartmentation necessitates the transport through intracellular membranes; members of the GLUT family and sodium-vitamin C cotransporters mediate the permeation of dehydroascorbic acid and ascorbate, respectively. Recent observations show that increased consumption and/or hindered entrance of ascorbate in/to a compartment results in pathological alterations partially resembling to scurvy, thus diseases of ascorbate compartmentation can exist. The review focuses on the reactions and transporters that can modulate ascorbate concentration and redox state in three compartments: endoplasmic reticulum, mitochondria and nucleus. By introducing the relevant experimental and clinical findings we make an attempt to coin the term of ascorbate compartmentation disease.

Transient knockdown of presenilin-1 provokes endoplasmic reticulum stress related formation of autophagosomes in HepG2 cells.

The involvement of presenilins in the endoplasmic reticulum (ER) related autophagy was investigated by their transient knockdown in HepG2 cells. The silencing of PSEN1 but not of PSEN2 led to cell growth impairment and decreased viability. PSEN1 silencing resulted in ER stress response as evidenced by the elevated levels of glucose regulated protein 78 (Grp78), protein disulfide isomerase (PDI), and CCAAT/enhancer-binding protein homologous protein (CHOP) and by the activation of activating transcription factor 6 (ATF6). The activation of autophagy was indicated by the increased procession of microtubule-associated light chain 3 protein isoform B (LC3B) and by decreased phosphorylation of mammalian target of rapamycin (mTOR) and 70kDa ribosomal protein S6 kinase (p70S6K). Formation of ER-related cytoplasmic vacuolization colocalizing with the autophagic marker LC3B was also observed. The morphological effects and LC3B activation in presenilin-1 knockdown cells could be prevented by using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin or by calcium chelation. The results show that presenilin-1 hampers the ER stress dependent initiation of macroautophagy.

Multiple roles of glucose-6-phosphatases in pathophysiology: state of the art and future trends.

BACKGROUND: The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. The enzyme is a part of a multicomponent system that includes several integral membrane proteins; the catalytic subunit (G6PC) and transporters for glucose-6-phosphate, inorganic phosphate and glucose. The G6PC gene family presently includes three members, termed as G6PC, G6PC2, and G6PC3. Although the three isoforms show a moderate amino acid sequence homology, their membrane topology and catalytic site are very similar. The isoforms are expressed differently in various tissues. Mutations in all three genes have been reported to be associated with human diseases. SCOPE OF REVIEW: The present review outlines the biochemical features of the G6PC gene family products, the regulation of their expression, their role in the human pathology and the possibilities for pharmacological interventions. MAJOR CONCLUSIONS: G6PCs emerge as integrators of extra- and intracellular glucose homeostasis. Beside the well known key role in blood glucose homeostasis, the members of the G6PC family seem to play a role as sensors of intracellular glucose and of intraluminal glucose/glucose-6-phosphate in the endoplasmic reticulum. GENERAL SIGNIFICANCE: Since mutations in the three G6PC genes can be linked to human pathophysiological conditions, the better understanding of their functioning in connection with genetic alterations, altered expression and tissue distribution has an eminent importance.

The glucose-6-phosphate transport is not mediated by a glucose-6-phosphate/phosphate exchange in liver microsomes.

A phosphate-linked antiporter activity of the glucose-6-phosphate transporter (G6PT) has been recently described in liposomes including the reconstituded transporter protein. We directly investigated the mechanism of glucose-6-phosphate (G6P) transport in rat liver microsomal vesicles. Pre-loading with inorganic phosphate (Pi) did not stimulate G6P or Pi microsomal inward transport. Pi efflux from pre-loaded microsomes could not be enhanced by G6P or Pi addition. Rapid G6P or Pi influx was registered by light-scattering in microsomes not containing G6P or Pi. The G6PT inhibitor, S3483, blocked G6P transport irrespectively of experimental conditions. We conclude that hepatic G6PT functions as an uniporter.

G6PT-H6PDH-11ßHSD1 triad in the liver and its implication in the pathomechanism of the metabolic syndrome.

The metabolic syndrome, one of the most common clinical conditions in recent times, represents a combination of cardiometabolic risk determinants, including central obesity, glucose intolerance, insulin resistance, dyslipidemia, non-alcoholic fatty liver disease and hypertension. Prevalence of the metabolic syndrome is rapidly increasing worldwide as a consequence of common overnutrition and consequent obesity. Although a unifying picture of the pathomechanism is still missing, the key role of the pre-receptor glucocorticoid activation has emerged recently. Local glucocorticoid activation is catalyzed by a triad composed of glucose-6-phosphate-transporter, hexose-6-phosphate dehydrogenase and 11ß-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum. The elements of this system can be found in various cell types, including adipocytes and hepatocytes. While the contribution of glucocorticoid activation in adipose tissue to the pathomechanism of the metabolic syndrome has been well established, the relative importance of the hepatic process is less understood. This review summarizes the available data on the role of the hepatic triad and its role in the metabolic syndrome, by confronting experimental findings with clinical observations.

Production of H2O2 in the endoplasmic reticulum promotes in vivo disulfide bond formation.

AIMS: Oxidative protein folding in the luminal compartment of endoplasmic reticulum (ER) is thought to be accompanied by the generation of H2O2, as side-product of disulfide bond formation. We aimed to examine the role of H2O2 produced in the lumen, which on one hand can lead to redox imbalance and hence can contribute to ER stress caused by overproduction of secretory proteins; on the other hand, as an excellent electron acceptor, H2O2 might serve as an additional pro-oxidant in physiological oxidative folding. RESULTS: Stimulation of H2O2 production in the hepatic ER resulted in a decrease in microsomal GSH and protein-thiol contents and in a redox shift of certain luminal oxidoreductases in mice. The oxidative effect, accompanied by moderate signs of ER stress and reversible dilation of ER cisternae, was prevented by concomitant reducing treatment. The imbalance also affected the redox state of pyridine nucleotides in the ER. Antibody producing cells artificially engineered with powerful luminal H2O2 eliminating system showed diminished secretion of mature antibody polymers, while incomplete antibody monomers/dimers were accumulated and/or secreted. INNOVATION: Evidence are provided by using in vivo models that hydrogen peroxide can promote disulfide bond formation in the ER. CONCLUSION: The results indicate that local H2O2 production promotes, while quenching of H2O2 impairs disulfide formation. The contribution of H2O2 to disulfide bond formation previously observed in vitro can be also shown in cellular and in vivo systems.

Expression of hexose-6-phosphate dehydrogenase in rat tissues.

Hexose-6-phosphate dehydrogenase (H6PD) is the main NADPH generating enzyme in the lumen of the endoplasmic reticulum. H6PD is regarded as an ancillary enzyme in prereceptorial glucocorticoid activation and probably acts as a nutrient sensor and as a prosurvival factor. H6PD expression was determined in a variety of rat and human tissues by detecting mRNA and protein levels, and by measuring its dehydrogenase and lactonase activities. It was found that H6PD was present in all investigated tissues; both expression and activity remained within an order of magnitude. Correlation was found between the dehydrogenase activity and protein or mRNA levels. The results confirmed the supposed housekeeping feature of the enzyme.

Contribution of fructose-6-phosphate to glucocorticoid activation in the endoplasmic reticulum: possible implication in the metabolic syndrome.

Both fructose consumption and increased intracellular glucocorticoid activation have been implicated in the pathogenesis of the metabolic syndrome. Glucocorticoid activation by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) depends on hexose-6-phosphate dehydrogenase (H6PD), which physically interacts with 11ß-HSD1 at the luminal surface of the endoplasmic reticulum (ER) membrane and generates reduced nicotinamide adenine dinucleotide phosphate for the reduction of glucocorticoids. The reducing equivalents for the reaction are provided by glucose-6-phosphate (G6P) that is transported by G6P translocase into the ER. Here, we show that fructose-6-phosphate (F6P) can substitute for G6P and is sufficient to maintain reductase activity of 11ß-HSD1 in isolated microsomes. Our findings indicate that the mechanisms of F6P and G6P transport across the ER membrane are distinct and provide evidence that F6P is converted to G6P in the ER lumen, thus yielding substrate for H6PD-dependent reduced nicotinamide adenine dinucleotide phosphate generation. Using the purified enzyme, we show that F6P cannot be directly dehydrogenated by H6PD, and we also excluded H6PD as a phosphohexose isomerase. Therefore, we postulate the existence of an ER luminal hexose-phosphate isomerase different from the cytosolic enzyme. The results suggest that cytosolic F6P promotes prereceptor glucocorticoid activation in white adipose tissue, which might have a role in the pathophysiology of the metabolic syndrome.

Hexose-6-phosphate dehydrogenase in the endoplasmic reticulum.

Hexose-6-phosphate dehydrogenase (H6PD) is a luminal enzyme of the endoplasmic reticulum that is distinguished from cytosolic glucose-6-phosphate dehydrogenase by several features. H6PD converts glucose-6-phosphate and NADP(+) to 6-phosphogluconate and NADPH, thereby catalyzing the first two reactions of the pentose-phosphate pathway. Because the endoplasmic reticulum has a separate pyridine nucleotide pool, H6PD provides NADPH for luminal reductases. One of these enzymes, 11beta-hydroxysteroid dehydrogenase type 1 responsible for prereceptorial activation of glucocorticoids, has been the focus of much attention as a probable factor in the pathomechanism of several human diseases including insulin resistance and the metabolic syndrome. This review summarizes recent advances related to the functions of H6PD.

Altered redox state of luminal pyridine nucleotides facilitates the sensitivity towards oxidative injury and leads to endoplasmic reticulum stress dependent autophagy in HepG2 cells.

Maintenance of the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum - an important pro-survival factor in the cell - is ensured by the concerted action of glucose-6-phosphate transporter and hexose-6-phosphate dehydrogenase. The mechanism by which the redox imbalance leads to cell death was investigated in HepG2 cells. The chemical inhibition of the glucose-6-phosphate transporter, the silencing of hexose-6-phosphate dehydrogenase and/or the glucose-6-phosphate transporter, or the oxidation of luminal NADPH by themselves did not cause a significant loss of cell viability. However, these treatments caused ER calcium store depletion. If these treatments were supplemented with the administration of a subliminal dose of the oxidizing agent menadione, endoplasmic reticulum vacuolization and a loss of viability were observed. Combined treatments resulted in the activation of ATF6 and procaspase-4, and in the induction of Grp78 and CHOP. In spite of the presence of UPR markers and proapoptotic signaling the effector caspases - caspase-3 and caspase-7 - were not active. On the other hand, an elevation of the autophagy marker LC3B was observed. Immunohistochemistry revealed a punctuated distribution of LC3B II, coinciding with the vacuolization of the endoplasmic reticulum. The results suggest that altered redox state of endoplasmic reticulum luminal pyridine nucleotides sensitizes the cell to autophagy.

Effect of nifedipine on capacitive calcium entry in Jurkat T lymphocytes.

The effect of nifedipine-an antagonist of L-type calcium (Ca(2+)) channels-on capacitative Ca(2+) entry (CCE) was studied in Jurkat T lymphocytes. CCE was induced by a variety of treatments each of which depleted intracellular Ca(2+) stores. Cells were treated with thapsigargin, ionomycin, anti-CD3 antibodies, and phytohaemagglutinin, or pre-incubated in a Ca(2+)-free medium. Activity of CCE was evaluated with a Ca(2+)-free/Ca(2+)-readmission protocol, in Fluo-3 pre-loaded cells. Nifedipine inhibited CCE in a dose-dependent manner. CCE inhibition was not due to non-specific effects on K(+) channels. Nifedipine, did not induce any membrane depolarization, as revealed by measurements of the plasma membrane potential with the fluorescent probe bis-oxonol. Moreover, experiments done under depolarizing conditions (i.e. by substituting Na(+) with K(+) ions in the medium) revealed that nifedipine could inhibit capacitative Ca(2+) entry independently of plasma membrane depolarization. We also demonstrated the presence in our Jurkat T-cells of transcripts for Ca(V)1.3 (alpha(1D)) and Ca(V)1.4 (alpha(1F)) L-type Ca(2+) channels. Verapamil and diltiazem, two unrelated blockers of L-type Ca(2+) channels, were less inhibitory on CCE. Possible mechanisms by which nifedipine interferes with Ca(2+) entry in these cells are discussed.

Hexose-6-phosphate dehydrogenase: linking endocrinology and metabolism in the endoplasmic reticulum.

Hexose-6-phosphate dehydrogenase (H6PD) got into the focus of interest due to its role in the prereceptorial activation of glucocorticoids, which has been implicated in the pathomechanism of metabolic syndrome. Genetic observations, results gained in H6PD knockout mice, and studies on differentiating adipocytes demonstrated the importance of the enzyme in metabolic regulation. A nutrient-sensing function can be postulated for the enzyme, which links metabolism to endocrinology in the endoplasmic reticulum. This review provides an overview of the recent developments concerning the enzyme and its impact on various branches of the intermediary metabolism, which make it an important subject for the research on obesity, diabetes, and metabolic syndrome.

Intraluminal hydrogen peroxide induces a permeability change of the endoplasmic reticulum membrane.

Gulonolactone treatment of mice resulted in the elevation of hepatic ascorbate and hydrogen peroxide levels accompanied by transient liver swelling and reversible dilatation of endoplasmic reticulum cisternae. Although a decrease in glutathione (reduced form)/total glutathione ratio was observed in microsomes, the redox state of luminal foldases remained unchanged and the signs of endoplasmic reticulum stress were absent. Increased permeability of the microsomal membrane to various compounds of low molecular weight was substantiated. It is assumed that Gulonolactone-dependent luminal hydrogen peroxide formation in the endoplasmic reticulum provokes a temporary increase in non-selective membrane permeability, which results in the dilation of the organelle and in enhanced transmembrane fluxes of small molecules.

Metyrapone prevents cortisone-induced preadipocyte differentiation by depleting luminal NADPH of the endoplasmic reticulum.

Preadipocyte differentiation is greatly affected by prereceptorial glucocorticoid activation catalyzed by 11beta-hydroxysteroid dehydrogenase type 1 in the lumen of the endoplasmic reticulum. The role of the local NADPH pool in this process was investigated using metyrapone as an NADPH-depleting agent. Metyrapone administered at low micromolar concentrations caused the prompt oxidation of the endogenous NADPH, inhibited the reduction of cortisone and enhanced the oxidation of cortisol in native rat liver microsomal vesicles. However, in permeabilized microsomes, it only slightly decreased both NADPH-dependent cortisone reduction and NADP(+)-dependent cortisol oxidation. Accordingly, metyrapone administration caused a switch in 11beta-hydroxysteroid dehydrogenase activity from reductase to dehydrogenase in both 3T3-L1-derived and human stem cell-derived differentiated adipocytes. Metyrapone greatly attenuated the induction of 11beta-hydroxysteroid dehydrogenase type 1 and the accumulation of lipid droplets during preadipocyte differentiation when 3T3-L1 cells were stimulated with cortisone, while it was much less effective in case of cortisol or dexamethasone. In conclusion, the positive feedback of glucocorticoid activation during preadipocyte differentiation is interrupted by metyrapone, which depletes NADPH in the endoplasmic reticulum. The results also indicate that the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum is important in the process of adipogenesis.

Constant expression of hexose-6-phosphate dehydrogenase during differentiation of human adipose-derived mesenchymal stem cells.

The reductase activity of 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) plays an important role in the growth and differentiation of adipose tissue via the prereceptorial activation of glucocorticoids. This enzyme colocalizes with hexose-6-phosphate dehydrogenase (H6PD) at the luminal surface of the endoplasmic reticulum membrane, and the latter enzyme provides NADPH to the former, which can thus act as an 11beta-reductase. It was suggested that, during adipogenesis, the increased expression of H6PD causes a dehydrogenase-to-reductase switch in the activity of HSD11B1. However, only the expression of the HSD11B1 has been extensively studied, and little is known about the expression of H6PD. Here, we investigated the expression and the activity of H6PD in the course of the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) and murine 3T3-L1 cells. It was found that H6PD is already present in adipose-derived stem cells and in 3T3-L1 fibroblasts even before the induction of adipogenesis. Moreover, mRNA and protein levels, as well as the microsomal H6PD activities remained unchanged during the differentiation. At the same time a great induction of HSD11B1 was observed in both cell types. The observed constant expression of H6PD suggests that HSD11B1 acts as a reductase throughout the adipogenesis process in human ADMSCs and murine 3T3-L1 cells.

Maintenance of luminal NADPH in the endoplasmic reticulum promotes the survival of human neutrophil granulocytes.

The present study demonstrates the expression of hexose-6-phosphate dehydrogenase and 11 beta-hydroxysteroid dehydrogenase type 1 in human neutrophils, and the presence and activity of these enzymes in the microsomal fraction of the cells. Their concerted action together with the previously described glucose-6-phosphate transporter is responsible for cortisone-cortisol interconversion detected in human neutrophils. Furthermore, the results suggest that luminal NADPH generation by the cortisol dehydrogenase activity of 11 beta-hydroxysteroid dehydrogenase type 1 prevents neutrophil apoptosis provoked by the inhibition of the glucose-6-phosphate transporter. In conclusion, the maintenance of the luminal NADPH pool is an important antiapoptotic factor in neutrophil granulocytes.