Promise(s) of Mesenchymal Stem Cells as an In Vitro Model System to Depict Pre-Diabetic/Diabetic Milieu in WNIN/GR-Ob Mutant Rats

Background

Development of model systems have helped to a large extent, in bridging gap to understand the mechanism(s) of disease including diabetes. Interestingly, WNIN/GR-Ob rats (Mutants), established at National Centre for Laboratory Animals (NCLAS) of National Institute of Nutrition (NIN), form a suitable model system to study obesity with Type 2 diabetes (T2D) demonstrating several secondary complications (cataract, cardiovascular complications, infertility, nephropathy etc). The present study has been carried out to explore the potent application(s) of multipotent stem cells such as bone marrow mesenchymal stem cells (BM-MSCs), to portray features of pre-diabetic/T2D vis-à-vis featuring obesity, with impaired glucose tolerance (IGT), hyperinsulinemia (HI) and insulin resistance (IR) seen with Mutant rats akin to human situation.

Methodology/Principal Findings

Primary cultures of BM-MSCs (third passage) from Mutants, its lean littermate (Lean) and parental control (Control) were characterized for: proliferation markers, disease memory to mark obesity/T2D/HI/IR which included phased gene expression studies for adipogenic/pancreatic lineages, inflammatory markers and differentiation ability to form mature adipocytes/Insulin-like cellular aggregates (ILCAs). The data showed that BM-MSCs from Mutant demonstrated a state of disease memory, depicted by an upregulated expression of inflammatory markers (IL-6, TNFα); increased stem cell recruitment (Oct-4, Sox-2) and proliferation rates (CD90+/CD29+, PDA, ‘S’ phase of cell cycle by FACS and BrdU incorporation); accelerated preadipocyte induction (Dact-1, PPARγ2) with a quantitative increase in mature adipocyte formation (Leptin); ILCAs, which were non-responsive to high glucose did confer the Obese/T2D memory in Mutants. Further, these observations were in compliance with the anthropometric data.

Conclusions

Given the ease of accessibility and availability of MSCs, the present study form the basis to report for the first time, application of BM-MSCs as a feasible in vitro model system to portray the disease memory of pre-clinical/T2D with IR - a major metabolic disorder of global concern.     

Functions of the ORF9-to-ORF12 gene cluster in varicella-zoster virus replication and in the pathogenesis of skin infection

The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKADelta10/11), ORF11 and ORF12 (POKADelta11/12), or ORF10, ORF11 and ORF12 (POKADelta10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKADelta11, POKADelta10/11, and POKADelta11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKADelta10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.     

Effect of IDA and TREN chelating agents and buffer systems on the purification of human IgG with immobilized nickel affinity membranes

The purification of IgG from human plasma was studied by comparing two affinity membranes complexed with Ni(II), prepared by coupling iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. The Ni(II)-TREN-PEVA hollow fiber membrane had lower capacity for human IgG than the complex Ni(II)-IDA-PEVA, but with similar selectivity. The IgG in peak fractions eluted from the Ni(II)-IDA-PEVA with a stepwise concentration gradient of Tris-HCl pH 7.0 (100-700 mM) reached a purity of 98% (based on IgG, IgM, IgA, albumin, and transferrin nephelometric analysis). Adsorption IgG data at different temperatures (4-37 degrees C) were analyzed using Langmuir model resulting in a calculated maximum capacity at 25 degrees C of 204.6 mg of IgG/g of dry membrane. Decrease in Kd with increasing temperature (1.7x10(-5) to 5.3x10(-6) M) indicated an increase in affinity with increased temperature. The positive value of enthalpy change (26.2 kJ/mol) indicated that the adsorption of IgG in affinity membrane is endothermic. Therefore, lower temperature induces adsorption as verified experimentally.     

New monolithic enzymatic micro-reactor for the fast production and purification of oligogalacturonides

Fast production and purification of alpha-(1,4)-oligogalacturonides was investigated using a new enzymatic reactor composed of a monolithic matrix. Pectin lyase from Aspergillus japonicus (Sigma) was immobilized on CIM-disk epoxy monolith. Studies were performed on free pectin lyase and immobilized pectin lyase to compare the optimum temperature, optimum pH, and thermal stability. It was determined that optimum temperature for free pectin lyase and immobilized pectin lyase on monolithic support is 30 degrees C, and optimum pH is 5. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method used for separation and purification of biomolecules due to high mass transfer rate. In this context, online one step production and purification of oligogalacturonides was investigated associating CIM-disk pectin lyase and CIM-disk DEAE. This efficient enzymatic bioreactor production of uronic oligosaccharides from polygalacturonic acid (PGA) constitutes an original fast process to generate bioactive oligouronides.     

Pregnane glycosides from Caralluma adscendens var. fimbriata

Eleven novel pregnane glycosides, 2-7 and 9-13, of which four, i.e., 10-13, comprised a new pregnane-type genin exhibiting a hydroxymethylene instead of a Me group at C(19), and the known pregnane glycoside stalagmoside V (8) were isolated from whole plants of Caralluma adscendens var. fimbriata, a native Indian succulent plant. Their structures were elucidated by extensive 2D-NMR spectroscopic studies.     

Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion

The mitochondrial permeability transition (MPT) plays an important role in hepatocyte death caused by ischemia-reperfusion (IR). This study investigated whether activation of the cellular oxygen-sensing signal cascade by prolyl hydroxylase inhibitors (PHI) protects against the MPT after hepatic IR. Ethyl 3,4-dihyroxybenzoate (EDHB, 100 mg/kg ip), a PHI, increased mouse hepatic hypoxia-inducible factor-1alpha and heme oxygenase-1 (HO-1). EDHB-treated and untreated mice were subjected to 1 h of warm ischemia to approximately 70% of the liver followed by reperfusion. Mitochondrial polarization, cell death, and the MPT were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. EDHB largely blunted alanine aminotransferase (ALT) release and necrosis after reperfusion. In vehicle-treated mice at 2 h after reperfusion, viable cells with depolarized mitochondria were 72%, and dead cells were 2%, indicating that depolarization preceded necrosis. Mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. NIM811, a specific inhibitor of the MPT, blocked mitochondrial depolarization after IR, further confirming that mitochondrial depolarization was due to MPT onset. EDHB decreased mitochondrial depolarization to 16% and prevented the MPT. Tin protoporphyrin (10 micromol/kg sc), an HO-1 inhibitor, partially abrogated protection by EDHB against ALT release, necrosis, and mitochondrial depolarization. In conclusion, IR causes the MPT and mitochondrial dysfunction, leading to hepatocellular death. PHI prevents MPT onset and liver damage through an effect mediated partially by HO-1.     

Enzyme field effect transistor (ENFET) for estimation of triglycerides using magnetic nanoparticles

Ion-selective field effect transistor (ISFET) is a robust platform to develop biosensors. A variety of methods are used including covalent attachment or polymer entrapment, to associate enzymes or antibodies to the gate surface of a FET. We have employed a novel method of retaining the enzyme molecules at the gate surface by immobilizing the enzyme on magnetic nickelferrite nanoparticles and applying a permanent magnet below the gate of the FET. We were able to estimate the triglyceride concentrations in the range of 0.1–1.5% by immobilizing a thermostable lipase on nanoparticles. Tributyrin, trioctanoate and triolein have given similar results. The reaction volume could be scaled down to 0.2 ml without a loss in slope or sensitivity. Ionic strength (>150 mM NaCl) has a strong influence on the sensitivity of the measurement. The advantages of this configuration of enzyme biosensor are reduction of mass transfer problems, increasing the amount of enzyme at the gate surface besides providing an opportunity to use a single FET device for multiple analyte detection.     

O(2)-sensing signal cascade: clamping of O(2) respiration, reduced ATP utilization, and inducible fumarate respiration

These studies explore the consequences of activating the prolyl hydroxylase (PHD) O(2)-sensing pathway in spontaneously twitching neonatal cardiomyocytes. Full activation of the PHD pathway was achieved using the broad-spectrum PHD inhibitor (PHI) dimethyloxaloylglycine (DMOG). PHI treatment of cardiomyocytes caused an 85% decrease in O(2) consumption and a 300% increase in lactic acid production under basal conditions. This indicates a approximately 75% decrease in ATP turnover rate, inasmuch as the increased ATP generation by glycolysis is inadequate to compensate for the lower respiration. To determine the extent to which decreased ATP turnover underlies the suppressed O(2) consumption, mitochondria were uncoupled with 2,4-dinitrophenol. We were surprised to find that 2,4-dinitrophenol failed to increase O(2) consumption by PHI-treated cells, indicating that electron transport chain activity, rather than ATP turnover rate, limits respiration in PHI-treated cardiomyocytes. Silencing of hypoxia-inducible factor-1alpha (HIF-1alpha) expression restored the ability of uncoupled PHI-treated myocytes to increase O(2) consumption; however, basal O(2) uptake rates remained low because of the unabated suppression of cellular ATP consumption. Thus it appears that respiration is actively "clamped" through an HIF-dependent mechanism, whereas HIF-independent mechanisms are responsible for downregulation of ATP consumption. In addition, we find that PHD pathway activation enables mitochondria to utilize fumarate as a terminal electron acceptor when cytochrome c oxidase is inactive. The source of fumarate for this unusual respiration is derived from aspartate via the purine nucleotide cycle. In sum, these studies show that the O(2)-sensing pathway is sufficient to actively "clamp" O(2) consumption and independently suppress cellular ATP consumption. The PHD pathway also enables the mitochondria to utilize fumarate for respiration.     

Biophysical analyses of human resistin: oligomer formation suggests novel biological function

Resistin, a small secreted peptide initially identified as a link between obesity and diabetes in mice, was shown to be involved in mediating inflammation in humans. We had shown earlier that recombinant human resistin has a tendency to form aggregates by formation of inter/intramolecular disulfide linkages and that it undergoes a concentration-dependent conformational change in secondary structure from alpha-helical to beta-sheet form. Here we report that this change in secondary structural conformation is due to the increase in the oligomeric form of human resistin as a function of protein concentration. Gel filtration analysis under different conditions further demonstrated that recombinant human resistin exists as a mixture of oligomer and trimer but is converted to a mixture of monomer and oligomer in the presence of 100 mM NaCl. We show that while the trimeric form of human resistin is stable to urea-induced denaturation, it is highly susceptible to NaCl and NaF, indicating the importance of ionic interactions in stabilization of trimer. In addition, urea was able to destabilize the oligomers indicating the involvement of hydrophobic interactions in oligomerization. Ionic as well as hydrophobic interactions stabilize the monomeric human resistin. Our data suggest that human resistin exists predominantly as oligomer and trimer in vitro. The oligomeric form of human resistin shows more potent effect on stimulation of proinflammatory cytokines. Therefore, it is very tempting to propose that the structural conformation of resistin may be involved in maintaining the very fine balance in regulation of macrophage function for successful response to a variety of pathological conditions.     

Protein adsorption on histidyl-aminohexyl-Sepharose 4B: I. Study of the mechanistic aspects of adsorption for the separation of human serum albumin from its non-enzymatic glycated isoforms (advanced glycosylated end products)

The characteristics of albumin adsorption on histidyl-aminohexyl-Sepharose 4B were investigated. In particular, the adsorption capacity of the gel was studied as a function of conductivity and pH of the running buffer. The adsorption was maximum at low salt concentration around neutral pH, involving electrostatic and hydrophobic interactions. Kinetic aspects were also investigated. Dissociation constant (K_D) and maximum capacity (Q_x) were, respectively, estimated to be 4.5×10⁻⁵ M (medium affinity) and 93.3 mg (high capacity) of human serum albumin per ml of adsorbent. According to these preliminary results, separation of HSA and its non-enzymatically glycated isoforms (conventionally named advanced glycated end products: AGEs) was achieved. Chromatographic potential of this separation tool is discussed.