Evaluation of Bone Markers in Type 2 Diabetes Mellitus.

BACKGROUND: Diabetes mellitus (DM) has affected over 387 million patients globally, expected to reach 592 million by the end of 2035. It is a metabolic disorder characterized by chronic hyperglycemia caused by either insulin deficiency, insulin resistance, or both. MATERIALS AND METHODS: The present study was designed to estimate the levels of different bone markers; serum Vitamin D, alkaline phosphatase, phosphorus, and calcium in patients with type 2 DM (T2DM). The study was conducted on patients aged 20-50 years diagnosed with T2DM, who were attending the outpatient/inpatient department of internal medicine. RESULTS: The levels of calcium were decreased in the patients with diabetes and also the study proved a negative correlation between calcium and random plasma glucose (RPG). There was a significant negative correlation between RPG and serum 25(OH)D3. CONCLUSION: We conclude that Vitamin D insufficiency is frequent in Moradabad, Uttar Pradesh. Sunshine exposure daily for 15 min on the face and hands is necessary to elevate the sunlight Vitamin D levels.

Résumé Contexte:Le diabète sucré (DM) a touché plus de 387 millions de patients dans le monde et devrait atteindre 592 millions d'ici la fin de l'année. 2035. Il s'agit d'un trouble métabolique caractérisé par une hyperglycémie chronique provoquée soit par un déficit en insuline, soit par une résistance à l'insuline, ou les deux.Matériels et méthodes:La présente étude a été conçue pour estimer les niveaux de différents marqueurs osseux; sérum Vitamine D, alkaline.Résultats:Les niveaux de calcium ont diminuéles patients diabétiques ainsi que l'étude ont prouvé une corrélation négative entre le calcium et le glucose plasmatique aléatoire (RPG). Il y avaitune corrélation négative significative entre le RPG et le sérum 25(OH)D3 phosphatase, phosphore et calcium chez les patients atteints de diabète de type 2 (DT2). L'étude a été menée sur des patients âgés de 20 à 50 ansdiagnostiqués atteints de DT2, qui fréquentaient le service de médecine interneambulatoire/hospitalisé.Conclusion:Nous concluons que l'insuffisance en vitamine D est fréquente chez Moradabad, Uttar Pradesh. Une exposition quotidienne au soleil pendant 15 minutes sur le visage et les mains est nécessaire pour élever les niveaux de vitamine D du soleil.

Chronic hyperglycemia impairs anti-microbial function of macrophages in response to Mycobacterium tuberculosis infection.

Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB), though the underlying mechanisms linking DM and TB remain ambiguous. Macrophages are a key player in the innate immune response and their phagocytic ability is enhanced in response to microbial infections. Upon infection or inflammation, they also repel invading pathogens by generating; reactive oxygen species (ROS), reactive nitrogen species (RNS), pro-inflammatory cytokines (IL-1ß and IL-6), and anti-inflammatory cytokines (IL-10). However, the robustness of these innate defensive capabilities of macrophages when exposed to hyperglycemia remains unclear. In our current work, we explored the production of these host defense molecules in response to challenge with Mycobacterium tuberculosis (Mtb) infection and lipopolysaccharide (LPS) stimulation. Utilizing peritoneal macrophages from high-fat diet + streptozotocin induced diabetic mice and hyperglycemic THP-1-derived macrophages as model systems; we found that LPS stimulation and Mtb infection were ineffective in stimulating the production of ROS, RNS, and pro-inflammatory cytokines in cells exposed to hyperglycemia. On the contrary, an increase in production of anti-inflammatory cytokines was observed. To confirm the mechanism of decreased anti-bacterial activity of the diabetic macrophage, we explored activation status of these compromised macrophages and found decreased surface expression of activation (TLR-4) and differentiation markers (CD11b and CD11c). We postulate that this could be the cause for higher susceptibility for Mtb infection among diabetic individuals.

Regulation of Macrophage Cell Surface GAPDH Alters LL-37 Internalization and Downstream Effects in the Cell.

Mycobacterium tuberculosis (M.tb), the major causative agent of tuberculosis, has evolved mechanisms to evade host defenses and persist within host cells. Host-directed therapies against infected cells are emerging as an effective option. Cationic host defense peptide LL-37 is known to internalize into cells and induce autophagy resulting in intracellular killing of M.tb. This peptide also regulates the immune system and interacts with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inside macrophages. Our investigations revealed that GAPDH moonlights as a mononuclear cell surface receptor that internalizes LL-37. We confirmed that the surface levels of purinergic receptor 7, the receptor previously reported for this peptide, remained unaltered on M.tb infected macrophages. Upon infection or cellular activation with IFNγ, surface recruited GAPDH bound to and internalized LL-37 into endocytic compartments via a lipid raft-dependent process. We also discovered a role for GAPDH in LL-37-mediated autophagy induction and clearance of intracellular pathogens. In infected macrophages wherein GAPDH had been knocked down, we observed an inhibition of LL-37-mediated autophagy which was rescued by GAPDH overexpression. This process was dependent on intracellular calcium and p38 MAPK pathways. Our findings reveal a previously unknown process by which macrophages internalize an antimicrobial peptide via cell surface GAPDH and suggest a moonlighting role of GAPDH in regulating cellular phenotypic responses of LL-37 resulting in reduction of M.tb burden.

Host glyceraldehyde-3-phosphate dehydrogenase-mediated iron acquisition is hijacked by intraphagosomal Mycobacterium tuberculosis.

Availability of iron is a key factor in the survival and multiplication of Mycobacterium tuberculosis (M.tb) within host macrophage phagosomes. Despite host cell iron regulatory machineries attempts to deny supply of this essential micronutrient, intraphagosomal M.tb continues to access extracellular iron. In the current study, we report that intracellular M.tb exploits mammalian secreted Glyceraldehyde 3-phosphate dehydrogenase (sGAPDH) for the delivery of host iron carrier proteins lactoferrin (Lf) and transferrin (Tf). Studying the trafficking of iron carriers in infected cells we observed that sGAPDH along with the iron carrier proteins are preferentially internalized into infected cells and trafficked to M.tb containing phagosomes where they are internalized by resident mycobacteria resulting in iron delivery. Collectively our findings provide a new mechanism of iron acquisition by M.tb involving the hijack of host sGAPDH. This may contribute to its successful pathogenesis and provide an option for targeted therapeutic intervention.

Exposure of a specific pleioform of multifunctional glyceraldehyde 3-phosphate dehydrogenase initiates CD14-dependent clearance of apoptotic cells.

Rapid clearance of apoptotic cells by phagocytes is crucial for organogenesis, tissue homeostasis, and resolution of inflammation. This process is initiated by surface exposure of various 'eat me' ligands. Though phosphatidylserine (PS) is the best recognized general recognition ligand till date, recent studies have shown that PS by itself is not sufficient for clearance of apoptotic cells. In this study, we have identified a specific pleioform of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) that functions as an 'eat me' signal on apoptotic cell surface. This specific form of GAPDH which is exposed on surface of apoptotic cells was found to interact with CD14 present on plasma membrane of phagocytes leading to their engulfment. This is the first study demonstrating the novel interaction between multifunctional GAPDH and the phagocytic receptor CD14 resulting in apoptotic cell clearance (efferocytosis).

Glyceraldehyde-3-Phosphate Dehydrogenase Facilitates Macroautophagic Degradation of Mutant Huntingtin Protein Aggregates.

Protein aggregate accumulation is a pathological hallmark of several neurodegenerative disorders. Autophagy is critical for clearance of aggregate-prone proteins. In this study, we identify a novel role of the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in clearance of intracellular protein aggregates. Previously, it has been reported that though clearance of wild-type huntingtin protein is mediated by chaperone-mediated autophagy (CMA), however, degradation of mutant huntingtin (mHtt with numerous poly Q repeats) remains impaired by this route as mutant Htt binds with high affinity to Hsc70 and LAMP-2A. This delays delivery of misfolded protein to lysosomes and results in accumulation of intracellular aggregates which are degraded only by macroautophagy. Earlier investigations also suggest that mHtt causes inactivation of mTOR signaling, causing upregulation of autophagy. GAPDH had earlier been reported to interact with mHtt resulting in cellular toxicity. Utilizing a cell culture model of mHtt aggregates coupled with modulation of GAPDH expression, we analyzed the formation of intracellular aggregates and correlated this with autophagy induction. We observed that GAPDH knockdown cells transfected with N-terminal mutant huntingtin (103 poly Q residues) aggregate-prone protein exhibit diminished autophagy. GAPDH was found to regulate autophagy via the mTOR pathway. Significantly more and larger-sized huntingtin protein aggregates were observed in GAPDH knockdown cells compared to empty vector-transfected control cells. This correlated with the observed decrease in autophagy. Overexpression of GAPDH had a protective effect on cells resulting in a decreased load of aggregates. Our results demonstrate that GAPDH assists in the clearance of protein aggregates by autophagy induction. These findings provide a new insight in understanding the mechanism of mutant huntingtin aggregate clearance. By studying the molecular mechanism of protein aggregate clearance via GAPDH, we hope to provide a new approach in targeting and understanding several neurodegenerative disorders.

Moonlighting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) modulates protein aggregation.

Onset of protein aggregation reflects failure of the cellular folding machinery to keep aggregation-prone protein from misfolding and accumulating into a non-degradable state. FRET based analysis and biochemical data reveal that cytosolic prion (cyPrP) and httQ-103 interact with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) leading to few detectable aggregates in GAPDH-over expressing cells.The preventive effect of GAPDH suggests that this abundant and long-lived cytoplasmic protein has an active role in the shielding and maintenance, in soluble form of proteins as heterogeneous as huntingtin and cyPrP.

Microbe-Derived Indole Metabolite Demonstrates Potent Multidrug Efflux Pump Inhibition in Staphylococcus aureus.

Efflux pumps are always at the forefront of bacterial multidrug resistance and account for the failure of antibiotics. The present study explored the potential of 2-(2-Aminophenyl) indole (RP2), an efflux pump inhibitor (EPI) isolated from the soil bacterium, to overcome the efflux-mediated resistance in Staphylococcus aureus. The RP2/antibiotic combination was tested against efflux pump over-expressed S. aureus strains. The compound was further examined for the ethidium bromide (EtBr) uptake and efflux inhibition assay (a hallmark of EPI functionality) and cytoplasmic membrane depolarization. The safety profile of RP2 was investigated using in vitro cytotoxicity assay and Ca2+ channel inhibitory effect. The in vivo efficacy of RP2 was studied in an animal model in combination with ciprofloxacin. RP2 exhibited the synergistic activity with several antibiotics in efflux pump over-expressed strains of S. aureus. In the mechanistic experiments, RP2 increased the accumulation of EtBr, and demonstrated the inhibition of its efflux. The antibiotic-EPI combinations resulted in extended post antibiotic effects as well as a decrease in mutation prevention concentration of antibiotics. Additionally, the in silico docking studies suggested the binding of RP2 to the active site of modeled structure of NorA efflux pump. The compound displayed low mammalian cytotoxicity and had no Ca2+ channel inhibitory effect. In ex vivo experiments, RP2 reduced the intracellular invasion of S. aureus in macrophages. Furthermore, the RP2/ciprofloxacin combination demonstrated remarkable efficacy in a murine thigh infection model. In conclusion, RP2 represents a promising candidate as bacterial EPI, which can be used in the form of a novel therapeutic regimen along with existing and upcoming antibiotics, for the eradication of S. aureus infections.

Moonlighting Protein Glyceraldehyde-3-Phosphate Dehydrogenase: A Cellular Rapid-Response Molecule for Maintenance of Iron Homeostasis in Hypoxia.

BACKGROUND/AIMS: Hypoxia triggers a rapid increase in iron demand to meet the requirements of enhanced erythropoiesis. The mobilization of iron stores from macrophage to plasma as holo-transferrin (Tf) from where it is accessible to erythroid precursor cells impacts iron homeostasis. Despite the immediate need for enhanced iron uptake by bone marrow cells, numerous studies have shown that transferrin receptor levels do not rise until more than 24 hours after the onset of hypoxia, suggesting the existence of heretofore unknown rapid response cellular machinery for iron acquisition in the early stages of cellular hypoxia. METHODS: We performed flow cytometry to measure cell surface levels of TfR1, GAPDH, and Tf binding after hypoxia treatment. We utilized FRET analysis and co-immunoprecipitation methods to establish the interaction between Tf and GAPDH. RESULTS: In the current study, we demonstrated that hypoxia induces K562 cells to translocate the cytosolic moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) onto cell surfaces and into the extracellular milieu to acquire transferrin-bound iron, even while levels of the classical transferrin receptor TfR1 (CD71) remain suppressed. GAPDH knockdown confirmed this protein's role in transferrin acquisition. Interestingly, macrophages did not show enhanced levels of extracellular GAPDH under hypoxia. CONCLUSION: Our results suggest the role of GAPDH-mediated Tf uptake as a rapid response mechanism by which cells acquire iron during the early stages of hypoxia. This is a tissue-specific phenomenon for the distinct requirements of cells that are consumers of iron versus cells that play a role in iron storage and recycling. This rapid deployment of an abundantly available multipurpose molecule allows hypoxic cells to internalize more Tf and maintain enhanced iron supplies in the early stages of hypoxia before specialized receptors can be synthesized and deployed to the cell membrane.

Trafficking of a multifunctional protein by endosomal microautophagy: linking two independent unconventional secretory pathways.

During physiologic stresses, like micronutrient starvation, infection, and cancer, the cytosolic moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is trafficked to the plasma membrane (PM) and extracellular milieu (ECM). Our work demonstrates that GAPDH mobilized to the PM, and the ECM does not utilize the classic endoplasmic reticulum-Golgi route of secretion; instead, it is first selectively translocated into early and late endosomes from the cytosol via microautophagy. GAPDH recruited to this common entry point is subsequently delivered into multivesicular bodies, leading to its membrane trafficking through secretion via exosomes and secretory lysosomes. We present evidence that both pathways of GAPDH membrane trafficking are up-regulated upon iron starvation, potentially by mobilization of intracellular calcium. These pathways also play a role in clearance of misfolded intracellular polypeptide aggregates. Our findings suggest that cells build in redundancy for vital cellular pathways to maintain micronutrient homeostasis and prevent buildup of toxic intracellular misfolded protein refuse.-Chauhan, A. S., Kumar, M., Chaudhary, S., Dhiman, A., Patidar, A., Jakhar, P., Jaswal, P., Sharma, K., Sheokand, N., Malhotra, H., Raje, C. I., Raje. M. Trafficking of a multifunctional protein by endosomal microautophagy: linking two independent unconventional secretory pathways.

Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.

Prokaryotic pathogens establish infection in mammals by capturing the proteolytic enzyme plasminogen (Plg) onto their surface to digest host extracellular matrix (ECM). One of the bacterial surface Plg receptors is the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In a defensive response, the host mounts an inflammatory response, which involves infiltration of leukocytes to sites of inflammation. This requires macrophage exit from the blood and migration across basement membranes, a phenomenon dependent on proteolytic remodeling of the ECM utilizing Plg. The ability of Plg to facilitate inflammatory cell recruitment critically depends on receptors on the surface of phagocyte cells. Utilizing a combination of biochemical, cellular, knockdown, and in vivo approaches, we demonstrated that upon inflammation, macrophages recruit GAPDH onto their surface to carry out the same task of capturing Plg to digest ECM to aid rapid phagocyte migration and combat the invading pathogens. We propose that GAPDH is an ancient, evolutionarily conserved receptor that plays a key role in the Plg-dependent regulation of macrophage recruitment in the inflammatory response to microbial aggression, thus pitting prokaryotic GAPDH against mammalian GAPDH, with both involved in a conserved role of Plg activation on the surface of their respective cells, to conflicting ends.-Chauhan, A. S., Kumar, M., Chaudhary, S., Patidar, A., Dhiman, A., Sheokand, N., Malhotra, H., Raje, C. I., Raje, M. Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.

Reverse overshot water-wheel retroendocytosis of apotransferrin extrudes cellular iron.

Iron (Fe), a vital micronutrient for all organisms, must be managed judiciously because both deficiency or excess can trigger severe pathology. Although cellular Fe import is well understood, its export is thought to be limited to transmembrane extrusion through ferroportin (also known as Slc40a1), the only known mammalian Fe exporter. Utilizing primary cells and cell lines (including those with no discernible expression of ferroportin on their surface), we demonstrate that upon Fe loading, the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is recruited to the cell surface, 'treadmills' apotransferrin in and out of the cell. Kinetic analysis utilizing labeled ligand, GAPDH-knockdown cells, (55)Fe-labeled cells and pharmacological inhibitors of endocytosis confirmed GAPDH-dependent apotransferrin internalization as a prerequisite for cellular Fe export. These studies define an unusual rapid recycling process of retroendocytosis for cellular Fe extrusion, a process mirroring receptor mediated internalization that has never before been considered for maintenance of cellular cationic homeostasis. Modulation of this unusual pathway could provide insights for management of Fe overload disorders.

Secreted multifunctional Glyceraldehyde-3-phosphate dehydrogenase sequesters lactoferrin and iron into cells via a non-canonical pathway.

Lactoferrin is a crucial nutritionally important pleiotropic molecule and iron an essential trace metal for all life. The current paradigm is that living organisms have evolved specific membrane anchored receptors along with iron carrier molecules for regulated absorption, transport, storage and mobilization of these vital nutrients. We present evidence for the existence of non-canonical pathway whereby cells actively forage these vital resources from beyond their physical boundaries, by secreting the multifunctional housekeeping enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the extracellular milieu. This effect's an autocrine/paracrine acquisition of target ligand into the cell. Internalization by this route is extensively favoured even by cells that express surface receptors for lactoferrin and involves urokinase plasminogen activator receptor (uPAR). We also demonstrate the operation of this phenomenon during inflammation, as an arm of the innate immune response where lactoferrin denies iron to invading microorganisms by chelating it and then itself being sequestered into surrounding host cells by GAPDH.