Withdrawal Of Glucose Research Articles

Unveiling Metabolic Vulnerability and Plasticity of Human Osteosarcoma Stem and Differentiated Cells to Improve Cancer Therapy.

Defining the metabolic phenotypes of cancer-initiating cells or cancer stem cells and of their differentiated counterparts might provide fundamental knowledge for improving or developing more effective therapies. In this context we extensively characterized the metabolic profiles of two osteosarcoma-derived cell lines, the 3AB-OS cancer stem cells and the parental MG-63 cells. To this aim Seahorse methodology-based metabolic flux analysis under a variety of conditions complemented with real time monitoring of cell growth by impedentiometric technique and confocal imaging were carried out. The results attained by selective substrate deprivation or metabolic pathway inhibition clearly show reliance of 3AB-OS on glycolysis and of MG-63 on glutamine oxidation. Treatment of the osteosarcoma cell lines with cisplatin resulted in additive inhibitory effects in MG-63 cells depleted of glutamine whereas it antagonized under selective withdrawal of glucose in 3AB-OS cells thereby manifesting a paradoxical pro-survival, cell-cycle arrest in S phase and antioxidant outcome. All together the results of this study highlight that the efficacy of specific metabolite starvation combined with chemotherapeutic drugs depends on the cancer compartment and suggest cautions in using it as a generalizable curative strategy.

Changes in Blood microRNA Expression and Early Metabolic Responsiveness 21 Days Following Bariatric Surgery.

Background: Early metabolic responses following bariatric surgery appear greater than expected given the initial weight loss and coincide with improvement in diabetes. We hypothesized that small non-coding microRNA changes might contribute to regulating mechanisms for metabolic changes and weight loss in patients with severe obesity and diabetes.Methods: Twenty-nine type 2 patients with severe obesity (mean BMI 46.2 kg/m2) and diabetes underwent Roux-en-Y gastric bypass (RYGB) surgery. Clinical measurements and fasting blood samples were taken preoperatively and at day 21 postoperatively. Normalization of fasting glucose and HbA1c following bariatric surgery (short-term diabetes remission) was defined as withdrawal of anti-diabetic medication and fasting glucose < 100 mg/dL (5.6 mmol/L) or HbA1c < 6.0%. MicroRNA expression was determined by quantitative polymerase chain reaction and tested for significant changes after surgery.Results: BMI decreased by 3.8 kg/m2 21 days postoperatively. Eighteen of 29 RYGB (62%) had short-term diabetes remission. Changes from pre- to post-surgery in 32 of 175 microRNAs were nominally significant (p < 0.05). Following multiple comparison adjustment, changes in seven microRNAs remained significant: miR-7-5p, let-7f-5p, miR-15b-5p, let-7i-5p, miR-320c, miR-205-5p, and miR-335-5p. Four pathways were over-represented by these seven microRNAs, including diabetes and insulin resistance pathways.Conclusion: Seven microRNAs showed significant changes 21 days after bariatric surgery. Functional pathways of the altered microRNAs were associated with diabetes-, pituitary-, and liver-related disease, with expression in natural killer cells, and pivotal intestinal pathology suggesting possible mechanistic roles in early diabetes responses following bariatric surgery.

A sodium-glucose cotransporter 2 inhibitor attenuates renal capillary injury and fibrosis by a vascular endothelial growth factor–dependent pathway after renal injury in mice

Multiple large clinical trials have shown that sodium-glucose cotransporter (SGLT) 2 inhibitors reduce the risk of renal events. However, the mechanism responsible for this outcome remains unknown. Here we investigated the effects of the SGLT2 inhibitor luseogliflozin on the development of renal fibrosis after renal ischemia/reperfusion injury in non-diabetic mice. Luseogliflozin significantly suppressed development of renal fibrosis, prevented peritubular capillary congestion/hemorrhage, attenuated CD31-positive cell loss, suppressed hypoxia, and increased vascular endothelial growth factor (VEGF)-A expression in the kidney after ischemia/reperfusion injury. Luseogliflozin failed to induce the above-mentioned protection in animals co-treated with sunitinib, a VEGF receptor inhibitor. Additionally, luseogliflozin reduced glucose uptake and increased VEGF-A expression in the kidneys of glucose transporter 2 (GLUT2)-downregulated mice following ischemia/reperfusion and in GLUT2-knock-down cells compared with those in normal controls. Withdrawal of glucose from cultured medium, to halt glucose uptake, remarkably increased VEGF-A expression and reversed the luseogliflozin-induced increase in VEGF-A expression in the proximal tubular cells. Thus, luseogliflozin prevented endothelial rarefaction and subsequent renal fibrosis after renal ischemia/reperfusion injury through a VEGF-dependent pathway induced by the dysfunction of proximal tubular glucose uptake in tubules with injury-induced GLUT2 downregulation.

Emerging Roles for Glycogen in the CNS.

The ability of glycogen, the depot into which excess glucose is stored in mammals, to act as a source of rapidly available energy substrate, has been exploited by several organs for both general and local advantage. The liver, expressing the highest concentration of glycogen maintains systemic normoglycemia ensuring the brain receives a supply of glucose in excess of demand. However the brain also contains glycogen, although its role is more specialized. Brain glycogen is located exclusively in astrocytes in the adult, with the exception of pathological conditions, thus in order to benefit neurons, and energy conduit (lactate) is trafficked inter-cellularly. Such a complex scheme requires cell type specific expression of a variety of metabolic enzymes and transporters. Glycogen supports neural elements during withdrawal of glucose, but once the limited buffer of glycogen is exhausted neural function fails and irreversible injury ensues. Under physiological conditions glycogen acts to provide supplemental substrates when ambient glucose is unable to support function during increased energy demand. Glycogen also supports learning and memory where it provides lactate to neurons during the conditioning phase of in vitro long-term potentiation (LTP), an experimental correlate of learning. Inhibiting the breakdown of glycogen or intercellular transport of lactate in in vivo rat models inhibits the retention of memory. Our current understanding of the importance of brain glycogen is expanding to encompass roles that are fundamental to higher brain function.

Modification of tumour cell metabolism modulates sensitivity to Chk1 inhibitor-induced DNA damage

Chk1 kinase inhibitors are currently under clinical investigation as potentiators of cytotoxic chemotherapy and demonstrate potent activity in combination with anti-metabolite drugs that increase replication stress through the inhibition of nucleotide or deoxyribonucleotide biosynthesis. Inhibiting other metabolic pathways critical for the supply of building blocks necessary to support DNA replication may lead to increased DNA damage and synergy with an inhibitor of Chk1. A screen of small molecule metabolism modulators identified combinatorial activity between a Chk1 inhibitor and chloroquine or the LDHA/LDHB inhibitor GSK 2837808A. Compounds, such as 2-deoxyglucose or 6-aminonicotinamide, that reduced the fraction of cells undergoing active replication rendered tumour cells more resistant to Chk1 inhibitor-induced DNA damage. Withdrawal of glucose or glutamine induced G1 and G2/M arrest without increasing DNA damage and reduced Chk1 expression and activation through autophosphorylation. This suggests the expression and activation of Chk1 kinase is associated with cells undergoing active DNA replication. Glutamine starvation rendered tumour cells more resistant to Chk1 inhibitor-induced DNA damage and reversal of the glutamine starvation restored the sensitivity of tumour cells to Chk1 inhibitor-induced DNA damage. Chk1 inhibitors may be a potentially useful therapeutic treatment for patients whose tumours contain a high fraction of replicating cells.

Selection of G1 Phase Yeast Cells for Synchronous Meiosis and Sporulation.

Centrifugal elutriation is a procedure that allows the fractionation of cell populations based upon their size and shape. This allows cells in distinct cell cycle stages can be captured from an asynchronous population. The technique is particularly helpful when performing an experiment to monitor the progression of cells through the cell cycle or meiosis. Yeast sporulation like gametogenesis in other eukaryotes initiates from the G1 phase of the cell cycle. Conveniently, S. cerevisiae arrest in G1 phase when starved for nutrients and so withdrawal of nitrogen and glucose allows cells to abandon vegetative growth in G1 phase before initiating the sporulation program. This simple starvation protocol yields a partial synchronization that has been used extensively in studies of progression through meiosis and sporulation. By using centrifugal elutriation it is possible to isolate a homogeneous population of G1 phase cells and induce them to sporulate synchronously, which is beneficial for investigating progression through meiosis and sporulation. An additionally benefit of this protocol is that cell populations can be isolated based upon size and both large and small cell populations can be tested for progression through meiosis and sporulation. Here we present a protocol for purification of G1 phase diploid cells for examining synchronous progression through meiosis and sporulation.

Analysis of the Cytotoxic Effects of Vitamin D3 on Colorectal, Breast and Cervical Carcinoma Cell Lines

Although evidences from epidemiological suggested possible involvement of vitamin D in the prevention and treatment of cancers, it is not fully known how vitamin D inhibits cancer cell growth. Recent studies have shown that 1,25-(OH)2D inhibits cancer cell proliferation by binding to vitamin-D receptor (VDR). The vitamin D - VDR complex in turn (a) upregulate cell cycle inhibitors p21 and p27; (b) promote apoptosis mediators caspase-3 and 7, Bad, p53 and PTEN; (c) arrest cells in senescence phase; (d) elevate cell differentiation; and (e) inhibit IGF signaling. Moreover, vitamin D reduces reactive oxygen species (ROS) there by prevents the progression of cancer cells. However, it is currently unknown whether vitamin D induced cancer cell death is mediated by its effect on ROS destroying Nrf2 signaling. In addition, it is also not known whether withdrawal of glucose improves the efficacy of vitamin D as presence of excess glucose promotes ROS in cancer cells. Therefore, first, efficacy of vitamin D for inhibiting the growth of cell lines HCT116, HeLa and MCF-7 was determined. Next, the effect of vitamin D on Nrf2 expression and activity in the presence and absence of glucose was assessed. The data showed that vitamin D inhibited the growth of HCT116, HeLa and MCF-7 cells in a dose dependent manner with more potency toward HCT116. Vitamin D reduced the levels of Nrf2 and NQO1 expression when HCT116 cells were treated in glucose lacking medium. But, despite a significant reduction in cell viability, no change in the Nrf2 expression was observed if the HCT-116 cells were treated with vitamin D dissolved in high glucose (4.5g/L) containing DMEM. Therefore, it is concluded that the cell growth inhibition by vitamin D, observed in the presence of glucose, is not at least mediated by Nrf2 modulation in HCT116 cells.

MYC-nick promotes cell migration by inducing fascin expression and Cdc42 activation

MYC-nick is a cytoplasmic, transcriptionally inactive member of the MYC oncoprotein family, generated by a proteolytic cleavage of full-length MYC. MYC-nick promotes migration and survival of cells in response to chemotherapeutic agents or withdrawal of glucose. Here we report that MYC-nick is abundant in colonic and intestinal tumors derived from mouse models with mutations in the Wnt, TGF-β, and PI3K pathways. Moreover, MYC-nick is elevated in colon cancer cells deleted for FBWX7, which encodes the major E3 ligase of full-length MYC frequently mutated in colorectal cancers. MYC-nick promotes the migration of colon cancer cells assayed in 3D cultures or grown as xenografts in a zebrafish metastasis model. MYC-nick accelerates migration by activating the Rho GTPase Cdc42 and inducing fascin expression. MYC-nick, fascin, and Cdc42 are frequently up-regulated in cells present at the invasive front of human colorectal tumors, suggesting a coordinated role for these proteins in tumor migration.

Index of Suspicion in The Nursery

A 4-day-old male infant presents in late November to the pediatrics clinic with decreased activity and poor feeding. The mother reports that he had been feeding and acting well until 1 day before presentation. The mother states that he has not had any rhinorrhea, nasal congestion, cough, vomiting, diarrhea, rash, or any atypical movements that could be seizures. She had not taken his temperature, and she states that he has not felt hot. The pediatrician becomes immediately concerned as she recognizes a lethargic infant in respiratory distress. A code is called, and the resuscitation is begun. The infant was a 3,083-g product of a 38+1 week gestation to a 28-year-old G2P1 woman. The pregnancy was only complicated by a positive group B streptococcus screen at 35 weeks' gestation and a history of herpes simplex virus in the past, but no lesions were noted before or during delivery. On presentation to labor and delivery, the fetus was noted to be tachycardic and the mother had mild abdominal tenderness, leading to initiation of ampicillin and gentamicin 16 hours before delivery. The duration of rupture of membranes was 4 hours. The infant was born via spontaneous vaginal delivery and was vigorous, with Apgar scores of 9 and 9 at 1 and 5 minutes. Because the mother was neither febrile nor tachycardic, it was decided that she did not have chorioamnionitis, so the infant received no further laboratory evaluation or antibiotics. The mother and infant were discharged from the hospital after 2 uneventful days. In the clinic, the 4-day-old infant has initial vital signs that included a temperature of 98.9°F, a heart rate of 170 beats per minute, a respiratory rate of 71 breaths per minute, an oxygen saturation (SaO2) of 80% on room air, and blood pressure 84/35 mm …

Effect of Varying Glucose Concentrations during In Vitro Maturation and Embryo Culture on Efficiency of In Vitro Embryo Production in Buffalo

This study was aimed to optimize glucose level at different stages of buffalo in vitro embryo production procedure. Three glucose levels (1.5, 5.6 and 10 mm) along with a control (0 mm) were used at three phases of in vitro fertilisation (IVF) procedure viz. in vitro maturation (IVM), in vitro culture (IVC-I) (12-72 hpi) and IVC-II (72 hpi to 7 dpi). Maturation rate of oocytes was found different under different glucose concentrations, and significantly more number of oocytes reached to MII under 5.6 mm glucose. The glucose levels at each phase (IVM, IVC-I and IVC-II) individually had significant effect on blastocyst rate, and the level used at one phase had significant effect on the outcome of next phase. Complete withdrawal of glucose from any of these stages irrespective of concentrations used at subsequent stage/s resulted in significantly lower number of blastocysts. However, the changing levels of glucose had differential effects during different phases of IVF steps. The most prominent effect of glucose level was observed during IVM. The presence of 5.6 mm glucose at all stages was most effective to yield highest blastocyst rate in buffalo IVF system.

Characterization of the Metabolic Phenotype of Rapamycin-Treated CD8+ T Cells with Augmented Ability to Generate Long-Lasting Memory Cells

BackgroundCellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized.Design and MethodsUsing lymphocytic choriomeningitis virus (LCMV) peptide gp33-specific CD8+ T cells derived from T cell receptor transgenic mice, we characterized the metabolic phenotype of proliferating T cells that were activated and expanded in vitro in the presence or absence of rapamycin, and determined the capability of these rapamycin-treated T cells to generate long-lived memory cells in vivo.ResultsAntigen-activated CD8+ T cells treated with rapamycin gave rise to 5-fold more long-lived memory T cells in vivo than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS). These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells.ConclusionsOur findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy.

The immature brain needs GABA to be excited and hyper-excited

The immature brain needs GABA to be excited and hyper-excited

Akt Requires Glucose Metabolism to Suppress Puma Expression and Prevent Apoptosis of Leukemic T Cells

The PI3K/Akt pathway is activated in stimulated cells and in many cancers to promote glucose metabolism and prevent cell death. Although inhibition of Akt-mediated cell survival may provide a means to eliminate cancer cells, this survival pathway remains incompletely understood. In particular, unlike anti-apoptotic Bcl-2 family proteins that prevent apoptosis independent of glucose, Akt requires glucose metabolism to inhibit cell death. This glucose dependence may occur in part through metabolic regulation of pro-apoptotic Bcl-2 family proteins. Here, we show that activated Akt relies on glycolysis to inhibit induction of Puma, which was uniquely sensitive to metabolic status among pro-apoptotic Bcl-2 family members and was rapidly up-regulated in glucose-deficient conditions. Importantly, preventing Puma expression was critical for Akt-mediated cell survival, as Puma deficiency protected cells from glucose deprivation and Akt could not readily block Puma-mediated apoptosis. In contrast, the pro-apoptotic Bcl-2 family protein Bim was induced normally even when constitutively active Akt was expressed, yet Akt could provide protection from Bim cytotoxicity. Up-regulation of Puma appeared mediated by decreased availability of mitochondrial metabolites rather than glycolysis itself, as alternative mitochondrial fuels could suppress Puma induction and apoptosis upon glucose deprivation. Metabolic regulation of Puma was mediated through combined p53-dependent transcriptional induction and control of Puma protein stability, with Puma degraded in nutrient-replete conditions and long lived in nutrient deficiency. Together, these data identify a key role for Bcl-2 family proteins in Akt-mediated cell survival that may be critical in normal immunity and in cancer through Akt-dependent stimulation of glycolysis to suppress Puma expression.

Spontaneous Network Events Driven by Depolarizing GABA Action in Neonatal Hippocampal Slices are Not Attributable to Deficient Mitochondrial Energy Metabolism

In two recent papers (Rheims et al., 2009; Holmgren et al., 2010), Zilberter and coworkers argue that the well known depolarizing GABA actions that take place at the cellular and network level in the neonatal hippocampus and neocortex in vitro are pathophysiological phenomena, attributable to deficient mitochondrial energy metabolism. In their experiments, supplementing the glucose-containing solution with weak-acid substrates of mitochondrial energy metabolism (such as β-hydroxy-butyrate, lactate, or pyruvate) abolished the spontaneous network events (giant depolarizing potentials; GDPs) and the underlying depolarizing actions of GABA. In this study, we made electrophysiological recordings of GDPs and monitored the mitochondrial membrane potential (Ψm) and intracellular pH (pH(i)) in CA3 neurons in neonatal rat hippocampal slices. Supplementing the standard physiological solution with l-lactate did not produce a change in Ψm, whereas withdrawal of glucose, in the presence or absence of l-lactate, was followed by a pronounced depolarization of Ψm. Furthermore, d-lactate (a poor substrate of mitochondrial metabolism) caused a prompt inhibition in GDP frequency which was similar to the effect of l-lactate. The suppression of GDPs was strictly proportional to the fall in pH(i) caused by weak carboxylic acids (l-lactate, d-lactate, or propionate) or by an elevated CO(2). The main conclusions of our work are that the inhibitory effect of l-lactate on GDPs is not mediated by mitochondrial energy metabolism, and that glucose at its standard 10 mm concentration is an adequate energy substrate for neonatal neurons in vitro. Notably, changes in pH(i) appear to have a very powerful modulatory effect on GDPs.

Abstract B26: Targeting glucose and glutamine metabolism in melanoma cells influences Noxa and Mcl-1 levels as well as susceptibility to TRAIL-induced apoptosis

Abstract Melanoma cells are notoriously resistant to conventional therapeutics (some of which trigger the intrinsic mitochondrial-based apoptotic pathway), whilst some melanoma cells are also resistant to extrinsic death ligand-mediated apoptosis. Emerging studies focused on metabolomics of cancer cells revealed that metabolic alterations in tumors could be exploited as novel targets for cancer therapy. Many tumor cells not only display a high rate of glucose uptake and glycolysis, but also exhibit increased glutamine consumption. In this study, apoptotic susceptibility to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) was investigated in four human melanoma cell lines in the presence of a metabolic inhibitor of glucose (2-deoxyglucose, 2DG, 10 mM) or glutamine (aminooxyacetate; AOA; 1mM, inhibitor of glutamate dependent transaminase). 2DG or AOA alone did not trigger significant apoptosis measured by Annexin V-PI staining and FACS analysis in these melanoma cell lines within a period of 24 hrs. However, the combined treatment with either 2DG plus TRAIL (100ng/ml), or AOA plus TRAIL, significantly induced apoptosis in 3 of 4 melanoma cell lines, compared to a single treatment protocol. Furthermore, the co-treatment selectively enhanced TRAIL mediated cytotoxicity only in melanoma cells, but not in normal human melanocytes or skin derived dermal fibroblasts. To more firmly establish the vital links between inhibition of glucose or glutamine metabolism and TRAIL efficacy, melanoma cells were also cultured with medium depleted of either glucose or glutamine for 1 day followed by adding TRAIL for another 24 hrs. Withdrawal of either glucose or glutamine by itself induced low level of spontaneous apoptosis in both melanoma cell lines tested. Similar to the treatment with 2DG or AOA, withdrawal of glucose or glutamine also sensitized both melanoma cell lines to TRAIL mediated apoptosis. The apoptosis induced by co-treatment with 2DG or AOA plus TRAIL was markedly blocked by a pan caspase inhibitor (Z-VAD), implicating a caspase-dependency. Western blotting analysis confirmed cotreatments effectively induced caspases 3 cleavage and Bid degradation in melanoma cells. Additionally 2DG or AOA plus TRAIL also resulted in loss of mitochondrial membrane potential (reduced Rhodamine123 uptake). Furthermore, both 2DG and AOA changed the balance between anti- and pro- apoptotic proteins of Bcl-2 family members. AOA was a stronger inducer compared to 2DG in enhancing BH3-only protein Noxa levels. 2DG was more effective in reducing Bcl-XL levels, while AOA more strongly inhibited Mcl-1 expression. Taken together, the findings of this study described a novel strategy to overcome the apoptotic resistance of melanoma cells by potentially activating both TRAIL-based, and mitochondrial-based, apoptosis by targeting either glucose or glutamine metabolic pathways for future melanoma therapeutic trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B26.

Relationship of Deregulated Signaling Converging onto mTOR with Prognosis and Classification of Lung Adenocarcinoma Shown by Two Independent In silico Analyses

There is marked disparity with a slight overlap among prognosis-predictive signatures reported thus far for lung cancers. In this study, we aimed at linking poor prognosis with particular pathways and/or functions of the gene sets involved to better understand the underlying molecular characteristics associated with the prognosis of lung adenocarcinomas. Gene set enrichment analysis identified a gene set down-regulated by rapamycin as the most significant, whereas several others responsive to withdrawal of glucose or amino acids, which are related to signaling converging onto mammalian target of rapamycin (mTOR), were also shown to be significantly associated, in addition to those related to DNA damage response and cell cycle progression. We also used connectivity map (C-MAP) analysis, an independent bioinformatics approach, to search for Food and Drug Administration-approved drugs that potentially transform an unfavorable signature to a favorable one. Those results identified inhibitors of phosphatidylinositol 3-kinase (PI3K) and mTOR, as well as unexpected drugs such as phenothiazine antipsychotics and resveratrol as potential candidates. Experimental validation revealed that the latter unexpected agents also inhibited signaling converging onto mTOR and exhibited antitumor activities. In addition, deregulation of multiple signaling converging onto mTOR was shown to be significantly associated with sensitivity to PI-103, a dual specificity PI3K/mTOR inhibitor that is not contained in the C-MAP database, lending further support for the connection. Our results clearly show the existence of gene set-definable, intrinsic heterogeneities in lung adenocarcinomas, which seem to be related to both clinical behavior and sensitivity to agents affecting the identified pathways.

Different states of energy metabolism in the vertebrate retina can be identified by stimulus-related changesin near UV transmission

Our aim was to identify different states of energy metabolism in the perfused isolated vertebrate retina by simultaneous recordings of light-evoked changes of transretinal potential (TRP) and spectroscopic changes related to oxidation of pyridine nucleotides. Isolated retinas were obtained from R.esc. and superfused. For each setting, three experiments were performed. Eighteen retinas were used. TRP as a response to light stimulation was recorded simultaneously with stimulus-induced transmission shift at 350 nm. These responses were recorded under normal conditions, under withdrawal of oxygen and glucose, and after addition of electron transport inhibitors and uncouplers of oxidative phosphorylation. Under normal conditions, TRP was strongly correlated with transmission shift after light stimulation. Without oxygen, amplitudes of stimulus-related TRP decreased and response related increase of transmission at 350 nm was reduced. The retina showed a much higher general absorption of the analyzing light. During glucose lack, an overall increase in transmission at 350 nm could be observed. The addition of Amytal yielded a specific reduction of the OFF components. Cyanide attenuated the ON and OFF components. Dinitrophenol yielded a considerable loss of the electrical ON response. Transmission change was affected less than electrical signal. Chlorophenylhydrazine induced a reproducible reduction of electrical responses, with a relative increase of transmission shift at 350 nm. In the isolated perfused retina, different states of energy metabolism and their specific impact on neuroretinal responses can be examined by simultaneous recordings of stimulus-related neuronal activity and spectroscopic changes.

Two days of a very low calorie diet reduces endogenous glucose production in obese type 2 diabetic patients despite the withdrawal of blood glucose–lowering therapies including insulin

The mechanism of the blood glucose–lowering effect of a 2-day very low calorie diet (VLCD; 1890 kJ/d) in combination with the cessation of all blood glucose–lowering agents was studied in 12 (7 women, 5 men) obese (body mass index, 36.3 ± 1.0 kg/m 2 [mean ± SEM]) type 2 diabetic patients (age, 55 ± 4 years; HbA 1c, 7.3% ± 0.4%) undergoing insulin therapy. Endogenous glucose production (EGP) and whole body glucose disposal (6,6 2H 2-glucose), lipolysis ( 2H 5-glycerol), and substrate oxidation (indirect calorimetry) rates were measured before and after the intervention in basal and hyperinsulinemic conditions. After 2 days of a VLCD and discontinuation of all blood glucose–lowering therapies, fasting plasma glucose levels did not increase (11.3 ± 1.3 vs 10.3 ± 1.0 mmol/L). Basal EGP significantly declined (14.2 ± 1.0 to 11.9 ± 0.7 μmol/kg per minute; P = .009). Basal metabolic clearance rate of glucose and rate of basal lipolysis did not change. During hyperinsulinemia, EGP (5.5 ± 0.8 to 5.2 ± 0.5 μmol/kg per minute), whole body glucose disposal (12.1 ± 0.7 to 11.3 ± 1.0 μmol/kg per minute), the metabolic clearance rate of glucose, and the rate of lipolysis did not change after the 2-day intervention. Cessation of blood glucose–lowering therapy in combination with a 2-day VLCD does not lead to hyperglycemia and is associated with a reduction in basal EGP. Insulin-stimulated whole body glucose disposal did not improve, nor did insulin suppressibility of EGP and lipolysis.

Acute ischemic injury of astrocytes is mediated by Na-K-Cl cotransport and not Ca2+ influx at a key point in white matter development.

Cerebral palsy is a common birth disorder that frequently involves ischemic-type injury to developing white matter (WM). Dead glial cells are a common feature of this injury and here we describe a novel form of acute ischemic cell death in developing WM astrocytes. Ischemia, modeled by the withdrawal of oxygen and glucose, evoked [Ca2+]i increases and cell death in astrocytes in post-natal day 10 (P10) rat optic nerve (RON). Removing extracellular Ca2+ prevented increases in [Ca2+]i but increased the amount of cell death. Astrocytes showed rapid [Na+]i increases during ischemia and cell death was reduced to control levels by substitution of extracellular Na+ or Cl- or by perfusion with bumetanide, a selective Na-K-Cl cotransport (NKCC) blocker. Astrocytes showed marked swelling during ischemia in the absence of extracellular Ca2+, which was blocked by bumetanide. Raising the extracellular osmolarity to limit water uptake reduced ischemic astrocyte death to control levels. Ultrastructural examination showed that post-ischemic astrocytes had lost their processes and frequently were necrotic, effects partially prevented by bumetanide. At this point in development, therefore, NKCC activation in astrocytes during ischemia produces an osmo-regulatory challenge. Astrocytes can subsequently regulate their cell volume in a Ca2+-dependent fashion but this will require ATP hydrolysis and does not protect the cells against acute cell death.

Cultivation in Glucose-Deprived Medium Stimulates Mitochondrial Biogenesis and Oxidative Metabolism in HepG2 Hepatoma Cells

In order to test the hypothesis that an imbalance between energy requirement and energy supply regulates mitochondrial genes and ultimately mitochondrial biogenesis, energy supply was challenged in HepG2 cells by withdrawal of glucose from the culture medium, making the cells exclusively dependent on mitochondrial ATP production. Such cells showed a 2-fold increase of cytochrome c oxidase activity, elevated levels of mitochondrial DNA, mitochondrial DNA encoded mRNAs and proteins, as well as the nuclear encoded mitochondrial transcription factor A. Lactate production was significantly reduced and glutamine was consumed as an alternative substrate for oxidative metabolism. Long-term adapted cells formed exclusively monolayers, while they normally grow in multilayers forming tumor spheroids. Also, long-term adapted cells proliferated significantly faster. No differences for the ATP/ADP ratio were observed, indicating that this is not the primary signal initiating the adaptative processes. These results show that mitochondrial biogenesis and oxidative metabolism are stimulated in HepG2 cells grown in the absence of fermentable glucose, probably in order to compensate for the diminished supply of glycolytic ATP.