Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog

Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive ³H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the non-radioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive ³H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive ³H-2-deoxy-D-glucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed.     

Protonated structures of naturally occurring deoxyribonucleic acids and their interaction with berberine

Protonation-induced conformational changes in natural DNAs of diverse base composition under the influence of low pH, low temperature, and low ionic strength have been studied using various spectroscopic techniques. At pH3.40, 10mM [Na+], and at 5 degrees C, all natural DNAs irrespective of base composition adopted an unusual and stable conformation remarkably different from the canonical B-form conformation. This protonated conformation has been characterized to have unique absorption and circular dichroic spectral characteristics and exhibited cooperative thermal melting profiles with decreased thermal melting temperatures compared to their respective B-form counterparts. The nature of this protonated structure was further investigated by monitoring the interaction of the plant alkaloid, berberine that was previously shown from our laboratory to differentially bind to B-form and H(L)-form of poly[d(G-C)] [Bioorg. Med. Chem.2003, 11, 4861]. Binding of berberine to protonated conformation of natural DNAs resulted in intrinsic circular dichroic changes as well as generation of induced circular dichroic bands for the bound berberine molecule with opposite signs and magnitude compared with B-form structures. Nevertheless, the binding of the alkaloid to both the B and protonated forms was non-linear and non-cooperative as revealed from Scatchard plots derived from spectrophotometric titration data. Steady state fluorescence studies on the other hand showed remarkable increase of the rather weak intrinsic fluorescence of berberine on binding to the protonated structure compared to the B-form structure. Taken together, these results suggest that berberine can detect the formation of significant population of H(L)-form structures under the influence of protonation irrespective of heterogeneous base compositions in natural DNAs.     

Binding affinity and in vitro cytotoxicity of harmaline targeting different motifs of nucleic acids: An ultimate drug designing approach

The work focuses towards interaction of harmaline, with nucleic acids of different motifs by multispectroscopic and calorimetric techniques. Findings of this study suggest that binding constant varied in the order single‐stranded (ss) poly(A) > double‐stranded calf thymus (CT) DNA > double‐stranded poly(G)·poly(C) > clover leaf tRNA^Phe. Prominent structural changes of ss poly(A), CT DNA, and poly(G)· poly(C) with concomitant induction of optical activity in the bound achiral alkaloid molecule was observed, while with tRNA^Phe, very weak induced circular dichroism perturbation was seen. The interaction was predominantly exothermic, enthalpy driven, and entropy favored with CT DNA and poly(G)·poly(C), while it was entropy driven with poly(A) and tRNA^Phe. Intercalated state of harmaline inside poly(A), CT DNA, and poly(G)·poly(C) was shown by viscometry, ferrocyanide quenching, and molecular docking. All these findings unequivocally pointed out preference of harmaline towards ss poly(A) inducing self‐structure formation. Furthermore, harmaline administration caused a significant decrease in proliferation of HeLa and HepG₂ cells with GI₅₀ of 28μM and 11.2μM, respectively. Nucleic acid fragmentation, cellular ultramorphological changes, decreased mitochondrial membrane potential, upregulation of p53 and caspase 3, generation of reactive oxygen species, and a significant increase in the G₂/M population made HepG₂ more prone to apoptosis than are HeLa cells.     

WISP-2/CCN5 is involved as a novel signaling intermediate in phorbol ester-protein kinase Calpha-mediated breast tumor cell proliferation

PMA and active phorbol esters stimulate the proliferation of various tumor cells, including ER-positive human breast tumor cell lines. However, the specific signaling pathways involved in the PMA-induced mitogenic effect on breast tumor cells have not been fully elucidated. In the present study, we explored the mechanisms associated with the mitogenic influence of PMA on breast tumor cells. Following an acute exposure (i.e., within 2 to 6 h) to PMA (50 nM), a mitogenic effect was observed on WISP-2/CCN5-positive breast tumor cell lines, including MCF-7, ZR-75-1 and SKBR-3 cells, and induction of WISP-2/CCN5 mRNA expression paralleled the observed mitogenic proliferation. This effect was undetected in WISP-2/CCN5 negative MDA-MB-231 breast tumor cells or human mammary epithelial cells with or without ER-alpha transfection. The mitogenic effect of PMA was perturbed by short hairpin RNA (shRNA)-mediated inhibition of WISP-2/CCN5 signaling in MCF-7 cells. Moreover, the upregulation of WISP-2/CCN5 by PMA is not ER dependent but is instead mediated through a complex PKCalpha-MAPK/ERK and SAPK/JNK signaling pathway, which leads to growth stimulation of MCF-7 breast tumor cells. These series of experiments provide the first evidence that WISP-2/CCN5 is a novel signaling molecule that critically participates in the mitogenic action of PMA on noninvasive, WISP-2/CCN5-positive breast tumor cells through PKCalpha-dependent, multiple molecular signal transduction pathways.     

Release Behaviour of Propranolol HCl from Hydrophilic Matrix Tablets Containing Psyllium Powder in Combination with Hydrophilic Polymers

The objective of this study was to investigate the release behaviour of propranolol hydrochloride from psyllium matrices in the presence hydrophilic polymers. The dissolution test was carried out at pH 1.2 and pH 6.8. Binary mixtures of psyllium and hydroxypropyl methylcellulose (HPMC) used showed that an increase in the percentage of HPMC in the binary mixtures caused a significant decrease in the release rate of propranolol. Psyllium–alginate matrices produced lower drug release as compared to when the alginate was the matrix former alone. When sodium carboxy methyl cellulose (NaCMC) was incorporated into the psyllium, the results showed that matrices containing the ratio of psyllium–NaCMC in the 1:1 ratio are able to slow down the drug release significantly as compared to matrices made from only psyllium or NaCMC as retardant agent suggesting that there could be a synergistic effect between psyllium and NaCMC. The double-layered tablets showed that the psyllium and HPMC in the outer shell of an inner formulation of psyllium alone had the greatest effect of protecting the inner core and thus producing the lowest drug release (DE = 38%, MDT = 93 min). A significant decrease in the value of n in Q = kt ⁿ from 0.70 to 0.51 as the psyllium content was increased from 50 to 150 mg suggests that the presence of psyllium in HPMC matrices affected the release mechanism. Psyllium powder had the ability in the combination with other hydrophilic polymers to produce controlled release profiles. Care and consideration should as such be taken when formulating hydrophilic matrices in different combinations.     

Interaction of isoquinoline alkaloid palmatine with deoxyribonucleic acids: binding heterogeneity, and conformational and thermodynamic aspects

The binding heterogeneity, conformational aspects, and energetics of the interaction of the cytotoxic plant alkaloid palmatine have been studied with various natural and synthetic DNAs. The alkaloid binds to calf thymus and Escherichia coli DNA that have mixed AT and GC sequences in almost equal proportions with positive cooperativity, while, with Clostridium perfringens and Micrococcus lysodeikticus DNA with predominantly high AT and GC sequences, respectively, noncooperative binding was observed. On further investigation with synthetic DNAs, the binding was observed to be cooperative with polymers like poly(dA).poly(dT) and poly(dG).poly(dC) having poly(purine)poly(pyrimidine) sequences, while with polymers poly(dA-dT).poly(dA-dT), poly(dA-dC).poly(dG-dT) and poly(dG-dC).poly(dG-dC), which have alternating purine-pyrimidine sequences, a non-cooperative binding phenomenon was observed. This suggests the binding heterogeneity of palmatine to the two types of sequences of base pairs. Circular dichroism (CD) studies revealed that the binding induced conformational changes in all the DNAs, but more importantly, the bound alkaloid molecules acquired induced optical activity, and the extent was dependent on the AT content and showed AT base-pair specificity. Energetics of the interaction of the alkaloid studied by highly sensitive isothermal titration calorimetry revealed that the binding was in most cases exothermic and favored by both enthalpy and entropy changes, while, in the case of the homo and hetero AT polymers, the same was predominantly entropy-driven. This study defines base-pair-dependent heterogeneity, conformational aspects, and energetics of palmatine binding to DNA.