Glucose starvation-induced turnover of the yeast glucose transporter Hxt1

Background

The budding yeast Saccharomyces cerevisiae possesses multiple glucose transporters with different affinities for glucose that enable it to respond to a wide range of glucose concentrations. The steady-state levels of glucose transporters are regulated in response to changes in the availability of glucose. This study investigates the glucose regulation of the low affinity, high capacity glucose transporter Hxt1.

Methods and results

Western blotting and confocal microscopy were performed to evaluate glucose regulation of the stability of Hxt1. Our results show that glucose starvation induces endocytosis and degradation of Hxt1 and that this event requires End3, a protein required for endocytosis, and the Doa4 deubiquitination enzyme. Mutational analysis of the lysine residues in the Hxt1 N-terminal domain demonstrates that the two lysine residues, K12 and K39, serve as the putative ubiquitin-acceptor sites by the Rsp5 ubiquitin ligase. We also demonstrate that inactivation of PKA (cAMP-dependent protein kinase A) is needed for Hxt1 turnover, implicating the role of the Ras/cAMP-PKA glucose signaling pathway in the stability of Hxt1.

Conclusion and general significance

Hxt1, most useful when glucose is abundant, is internalized and degraded when glucose becomes depleted. Of note, the stability of Hxt1 is regulated by PKA, known as a positive regulator for glucose induction of HXT1 gene expression, demonstrating a dual role of PKA in regulation of Hxt1.     

High glucose induces vascular endothelial growth factor production in human synovial fibroblasts through reactive oxygen species generation

Background

Diabetes is an independent risk factor of osteoarthritis (OA). Angiogenesis is essential for the progression of OA. Here, we investigated the intracellular signaling pathways involved in high glucose (HG)-induced vascular endothelial growth factor (VEGF) expression in human synovial fibroblast cells.

Methods

HG-mediated VEGF expression was assessed with qPCR and ELISA. The mechanisms of action of HG in different signaling pathways were studied using Western blotting. Knockdown of proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the VEGF promoter.

Results

Stimulation of OA synovial fibroblasts (OASF) with HG induced concentration- and time-dependent increases in VEGF expression. Treatment of OASF with HG increased reactive oxygen species (ROS) generation. Pretreatment with NADPH oxidase inhibitor (APO or DPI), ROS scavenger (NAC), PI3K inhibitor (Ly294002 or wortmannin), Akt inhibitor, or AP-1 inhibitor (curcumin or tanshinone IIA) blocked the HG-induced VEGF production. HG also increased PI3K and Akt activation. Treatment of OASF with HG increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the VEGF promoter.

Conclusions

Our results suggest that the HG increases VEGF expression in human synovial fibroblasts via the ROS, PI3K, Akt, c-Jun and AP-1 signaling pathway.

General significance

We link high glucose on VEGF expression in osteoarthritis.     

Cloning,characterization, and expression of glucose transporter 2 in the freeze-tolerant wood frog, Rana sylvatica

Background

The essential role of glucose transporter 2 (GLUT2) in glucose homeostasis has been extensively studied in mammals; however, little is known about this important protein in lower vertebrates. The freeze-tolerant wood frog (Rana sylvatica), which copiously mobilizes glucose in response to freezing, represents an excellent system for the study of glucose transport in amphibians.

Methods

GLUT2 was sequenced from northern and southern phenotypes of R. sylvatica, as well as the freeze-intolerant Rana pipiens. These proteins were expressed and functionally characterized in Xenopus oocytes. Abundance of GLUT2 in tissues was analyzed using immunoblotting techniques.

Results

GLUT2s cloned from these anurans encoded proteins with high sequence homologies to known vertebrate GLUT2s and had similar transport properties, although, notably, transport of the glucose analog 3-O-methyl-d-glucose (3-OMG) was strongly inhibited by 150 mM urea. Proteins from all study subjects had similar affinity constants (~ 12 mM) and other kinetic properties; however, GLUT2 abundance in liver was 3.5-fold greater in northern R. sylvatica than in the southern conspecific and R. pipiens.

Conclusion

Our results indicate that amphibian GLUT2s are structurally and functionally similar to their homologs in other vertebrates, attesting to the conserved nature of this transport protein. The greater abundance of this protein in the northern phenotype of R. sylvatica suggests that these transporters contribute importantly to freezing survival.

General significance

This study provides the first functional characterization of any GLUT isoform from an anuran amphibian and novel insights into the role of these proteins in glucose homeostasis and cryoprotectant mobilization in freeze-tolerant vertebrates.     

Simple and robust strategy for potentiometric detection of glucose using fluorinated phenylboronic acid self-assembled monolayer

Background

Field effect transistor (FET) based signal-transduction (Bio-FET) is an emerging technique for label-free and real-time basis biosensors for a wide range of targets. Glucose has constantly been of interest due to its clinical relevance. Use of glucose oxidase (GOD) and a lectin protein Concanavalin A are two common strategies to generate glucose-dependent electrochemical events. However, these protein-based materials are intolerant of long-term usage and storage due to their inevitable denaturing.

Methods

A phenylboronic acid (PBA) modified self-assembled monolayer (SAM) on a gold electrode with an optimized disassociation constant of PBA, that is, 3-fluoro-4-carbamoyl-PBA possessing its pKa of 7.1, was prepared and utilized as an extended gate electrode for Bio-FET.

Results

The prepared electrode showed a glucose-dependent change in the surface potential under physiological conditions, thus providing a remarkably simple rationale for the glyco-sensitive Bio-FET. Importantly, the PBA modified electrode showed tolerance to relatively severe heat and drying treatments; conditions under which protein based materials would surely be denatured.

Conclusions

A PBA modified SAM with optimized disassociation constant (pKa) can exhibit a glucose-dependent change in the surface potential under physiological conditions, providing a remarkably simple but robust method for the glyco-sensing.

General significance

This protein-free, totally synthetic glyco-sensing strategy may offer cheap, robust and easily accessible platform that may be useful in developing countries. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

The AMPK-SIRT signaling network regulates glucose tolerance under calorie restriction conditions

Aims

SIRT1 and AMP-activated protein kinase (AMPK) share common activators, actions and target molecules. Previous studies have suggested that a putative SIRT1-AMPK regulatory network could act as the prime initial sensor for calorie restriction-induced adaptations in skeletal muscle—the major site of insulin-stimulated glucose disposal. Our study aimed to investigate whether a feedback loop exists between AMPK and SIRT1 in skeletal muscle and how this may be involved glucose tolerance.

Main methods

To investigate this, we used skeletal muscle-specific AMPKα1/2 knockout mice (AMPKα1/2^−/−) fed ad libitum (AL) or a 30% calorie restricted (CR) diet and L6 rat myoblasts incubated with SIRT1 inhibitor (EX527).

Key findings

CR-AMPKα1/2^−/− displayed impaired glucose tolerance (*p < 0.05), in association with down-regulated SIRT1 and PGC-1α expression (< 300% vs. CR-WT, ^±±p < 0.01). Moreover, AMPK activity was decreased following SIRT1 inhibition in L6 cells (~ 0.5-fold vs. control, *p < 0.05).

Significance

This study demonstrates that skeletal muscle-specific AMPK deficiency impairs the beneficial effects of CR on glucose tolerance and that these effects may be dependent on reduced SIRT1 levels.     

Depletion of casein kinase I leads to a NAD(P)/NAD(P)H balance-dependent metabolic adaptation as determined by NMR spectroscopy-metabolomic profile in Kluyveromyces lactis

Background

In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag⁻ phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing.

Methods

A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses.

Results

Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced.

Conclusions

The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)⁺/NAD(P)H redox balance state.

General significance

The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.     

Inorganic phosphate uptake in unicellular eukaryotes

Background

Inorganic phosphate (P_i) is an essential nutrient for all organisms. The route of P_i utilization begins with P_i transport across the plasma membrane.

Scope of review

Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of P_i transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding P_i uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum.

Major conclusion

P_i uptake is the key step of P_i homeostasis and in the subsequent signaling event in eukaryotic microorganisms.

General significance

Biochemical and structural studies are important for clarifying mechanisms of P_i homeostasis, as well as P_i sensor and downstream pathways, and raise possibilities for future studies in this field.     

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.     

Bioreactors to influence stem cell fate: Augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems

Background

Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro.

Scope of review

This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems.

Major conclusions

The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased.

General significance

Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Activation of H-ATPase by glucose in Saccharomyces cerevisiae involves a membrane serine protease

Background

Glucose induces H⁺-ATPase activation in Saccharomyces cerevisiae. Our previous study showed that (i) S. cerevisiae plasma membrane H⁺-ATPase forms a complex with acetylated tubulin (AcTub), resulting in inhibition of the enzyme activity; (ii) exogenous glucose addition results in the dissociation of the complex and recovery of the enzyme activity.

Methods

We used classic biochemical and molecular biology tools in order to identify the key components in the mechanism that leads to H⁺-ATPase activation after glucose treatment.

Results

We demonstrate that glucose-induced dissociation of the complex is due to pH-dependent activation of a protease that hydrolyzes membrane tubulin. Biochemical analysis identified a serine protease with a kDa of 35–40 and an isoelectric point between 8 and 9. Analysis of several knockout yeast strains led to the detection of Lpx1p as the serine protease responsible of tubulin proteolysis. When lpx1Δ cells were treated with glucose, tubulin was not degraded, the AcTub/H⁺-ATPase complex did not undergo dissociation, and H⁺-ATPase activation was significantly delayed.

Conclusion

Our findings indicate that the mechanism of H⁺-ATPase activation by glucose involves a decrease in the cytosolic pH and consequent activation of a serine protease that hydrolyzes AcTub, accelerating the process of the AcTub/H⁺-ATPase complex dissociation and the activation of the enzyme.

General significance

Our data sheds light into the mechanism by which acetylated tubulin dissociates from the yeast H⁺-ATPase, identifying a degradative step that remained unknown. This finding also proposes an indirect way to pharmacologically regulate yeast H⁺-ATPase activity and open the question about mechanistic similarities with other higher eukaryotes.     

β-Glucan from Saccharomyces cerevisiae reduces lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages

Background

β-Glucans obtained from fungi, such as baker's yeast (Saccharomyces cerevisiae)-derived β-glucan (BBG), potently activate macrophages through nuclear factor κB (NFκB) translocation and activation of its signaling pathways. The mechanisms by which β-glucans activate these signaling pathways differ from that of lipopolysaccharide (LPS). However, the effects of β-glucans on LPS-induced inflammatory responses are poorly understood. Here, we examined the effects of BBG on LPS-induced inflammatory responses in RAW264.7 mouse macrophages.

Methods

We explored the actions of BBG in RAW264.7 macrophages.

Results

BBG inhibited LPS-stimulated nitric oxide (NO) production in RAW264.7 macrophages by 35–70% at concentrations of 120–200 μg/ml. BBG also suppressed mRNA and protein expression of LPS-induced inducible NO synthase (iNOS) and mitogen-activated protein kinase phosphorylation, but not NFκB activation. By contrast, a neutralizing antibody against dectin-1, a β-glucan receptor, did not affect BBG-mediated inhibition of NO production. Meanwhile, BBG suppressed Pam3CSK-induced NO production. Moreover, BBG suppressed LPS-induced production of pro-and anti-inflammatory cytokines, including interleukin (IL)-1α, IL-1ra, and IL-27.

Conclusions

Our results indicate that BBG is a powerful inhibitor of LPS-induced NO production by downregulating iNOS expression. The mechanism involves inactivation of mitogen-activated protein kinase and TLR2 pathway, but is independent of dectin-1.

General significance

BBG might be useful as a novel agent for the chemoprevention of inflammatory diseases.     

3β-taraxerol of Mangifera indica, a PI3K dependent dual activator of glucose transport and glycogen synthesis in 3T3-L1 adipocytes

Background

The present study focuses on identifying and developing an anti-diabetic molecule from plant sources that would effectively combat insulin resistance through proper channeling of glucose metabolism involving glucose transport and storage.

Methods

Insulin-stimulated glucose uptake formed the basis for isolation of a bioactive molecule through column chromatography followed by its characterization using NMR and mass spectroscopic analysis. Mechanism of glucose transport and storage was evaluated based on the expression profiling of signaling molecules involved in the process.

Results

The study reports (i) the isolation of a bioactive compound 3β-taraxerol from the ethyl acetate extract (EAE) of the leaves of Mangifera indica (ii) the bioactive compound exhibited insulin-stimulated glucose uptake through translocation and activation of the glucose transporter (GLUT4) in an IRTK and PI3K dependent fashion. (iii) the fate of glucose following insulin-stimulated glucose uptake was ascertained through glycogen synthesis assay that involved the activation of PKB and suppression of GSK3β.

General significance

This study demonstrates the dual activity of 3β-taraxerol and the ethyl acetate extract of Mangifera indica as a glucose transport activator and stimulator of glycogen synthesis. 3β-taraxerol can be validated as a potent candidate for managing the hyperglycemic state.     

Sphingolipids and mitochondrial function in budding yeast

Background

Sphingolipids (SLs) are not only key components of cellular membranes, but also play an important role as signaling molecules in orchestrating both cell growth and apoptosis. In Saccharomyces cerevisiae, three complex SLs are present and hydrolysis of either of these species is catalyzed by the inositol phosphosphingolipid phospholipase C (Isc1p). Strikingly, mutants deficient in Isc1p display several hallmarks of mitochondrial dysfunction such as the inability to grow on a non-fermentative carbon course, increased oxidative stress and aberrant mitochondrial morphology.

Scope of review

In this review, we focus on the pivotal role of Isc1p in regulating mitochondrial function via SL metabolism, and on Sch9p as a central signal transducer. Sch9p is one of the main effectors of the target of rapamycin complex 1 (TORC1), which is regarded as a crucial signaling axis for the regulation of Isc1p-mediated events. Finally, we describe the retrograde response, a signaling event originating from mitochondria to the nucleus, which results in the induction of nuclear target genes. Intriguingly, the retrograde response also interacts with SL homeostasis.

Major conclusions

All of the above suggests a pivotal signaling role for SLs in maintaining correct mitochondrial function in budding yeast.

General significance

Studies with budding yeast provide insight on SL signaling events that affect mitochondrial function.     

Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells

Background

Intracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes.

Methods

Secretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy.

Results

Engineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi.

Conclusions

Generation of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved.

General significance

These results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues.