Ginsenosides Rb1 and Rg1 suppress triglyceride accumulation in 3T3-L1 adipocytes and enhance beta-cell insulin secretion and viability in Min6 cells via PKA-dependent pathways

Ginseng root is known to induce anti-diabetic activity, but the key components involved are unknown. We investigated which major ginsenosides in ginseng enhanced glucose homeostasis by in vitro studies. Rb1 and Rg1 reduced the triglyceride accumulation in 3T3-L1 adipocytes by activating PKA with increased intracellular cAMP. However, the insulin-stimulated glucose uptake was enhanced by Rb1 and Rg1 via activation of phosphatidylinositol-3 kinase. Rb1 and Rg1 promoted glucose-stimulated insulin secretion and cell viability in Min6 cells through PKA which augmented IRS2 expression to enhance insulin/IGF-1 signaling. These results suggest that Rb1 and Rg1 improved glucose homeostasis through the activation of a PKA like glucagon-like peptide-1 receptor agonist.     

SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expression

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.     

Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells

Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-l-cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.     

A role of hexokinases in plant resistance to oxidative stress and pathogen infection

Previously, we reported that mitochondria-associated hexokinases are active in controlling programmed cell death in plants (Plant Cell 18, 2341-2355). Here, we investigated their role under abiotic- and biotic-stress conditions. Expression ofNbHxk1, aNicotiana benthamiana hexokinase gene, was stimulated by treatment with salicylic acid or methyl viologen (MV), and was also up-regulated by pathogen infection. In response to MV-induced oxidative stress, NbHxk1-silenced plants exhibited increased susceptibility, while the HXK1— and HXK2-overexpressingArabidopsis plants had enhanced tolerance. Moreover, those overexpressing plants showed greater resistance to the necrotrophic fungal pathogenAlternaria brassicicola. HXK-over-expression also mildly protected plants against the bacterial pathogenPseudomonas syringae pv.tomato DC3000, a response that was accompanied by increased H₂O₂ production and elevatedPR1 gene expression. These results demonstrate that higher levels of hexokinase confer improved resistance to MV-induced oxidative stress and pathogen infection.     

TSCOT+ thymic epithelial cell-mediated sensitive CD4 tolerance by direct presentation

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.     

Time-course Changes in Immunoreactivities of Glucokinase and Glucokinase Regulatory Protein in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia–reperfusion (I–R). Five days after I–R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.     

Reprofiling of pyrimidine-based DAPK1/CSF1R dual inhibitors: identification of 2,5-diamino-4-pyrimidinol derivatives as novel potential anticancer lead compounds

Abstract

Hybridization of reported weakly active antiproliferative hit 5-amino-4-pyrimidinol derivative with 2-anilino-4-phenoxypyrimidines suggests a series of 2,5-diamino-4-pyrimidinol derivatives as potential antiproliferative agents. Few compounds belonging to the proposed series were reported as CSF1R/DAPK1 inhibitors as anti-tauopathies. However, the correlation between CSF1R/DAPK1 signalling pathways and cancer progression provides motives to reprofile them against cancer therapy. The compounds were synthesised, characterized, and evaluated against M-NFS-60 cells and a kinase panel which bolstered predictions of their antiproliferative activity and suggested the involvement of diverse molecular targets. Compound 6e, the most potent in the series, showed prominent broad-spectrum antiproliferative activity inhibiting the growth of hematological, NSCLC, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers by 84.1, 52.79, 72.15, 66.34, 66.48, 51.55, 55.95, 61.85, and 60.87%, respectively. Additionally, it elicited an IC₅₀ value of 1.97?µM against M-NFS-60 cells and good GIT absorption with P_e value of 19.0?±?1.1?×?10^?6?cm/s (PAMPA-GIT). Molecular docking study for 6e with CSF1R and DAPK1 was done to help to understand the binding mode with both kinases. Collectively, compound 6e could be a potential lead compound for further development of anticancer therapies.     

Myocardial fibrosis, impaired coronary hemodynamics, and biventricular dysfunction in salt-loaded SHR

Arterial pressure in most experimental and clinical hypertensions is exacerbated by salt. The effects of salt excess on right and left ventricular (RV and LV, respectively) functions and their respective coronary vasodilatory responses have been less explored. We therefore examined the effects of 8 wk of NaCl excess (8% in food) on arterial pressure, RV and LV functions (maximal rate of increase and decrease of ventricular pressure; dP/dt(max) and dP/dt(min)), coronary hemodynamics (microspheres), and collagen content (hydroxyproline assay and collagen volume fraction) in young adult normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR), aged 16 wk by the end of the study. Prolonged salt excess in WKY and SHR elevated pressure only modestly, but it markedly increased LV mass, especially in SHR. Moreover, salt excess significantly impaired RV and LV diastolic function in SHR but only LV diastolic function in WKY rats. However, salt loading affected neither RV nor LV contractile function in both strains. Interstitial and perivascular collagen deposition was increased, whereas coronary vasodilatory responses to dipyridamole diminished in both ventricles in the salt-loaded SHR but not in WKY rats. Therefore, accumulation of ventricular collagen as well as altered myocardial perfusion importantly contributed to the development of salt-related RV and LV dysfunctions in this model of naturally occurring hypertension. The unique effects of salt loading on both ventricles in SHR, but not WKY rats, strongly suggest that nonhemodynamic mechanisms in hypertensive disease participate pathophysiologically with salt-loading hypertension. These findings point to the conclusion that the concept of "salt sensitivity" in hypertension is far more complex than simply its effects on arterial pressure or the LV.     

Effect of JGK-263 as a new glycogen synthase kinase-3β inhibitor on extrinsic apoptosis pathway in motor neuronal cells

Glycogen synthase kinase-3β (GSK-3β) has been identified as one of the important pathogenic mechanisms in motor neuronal death. GSK-3β inhibitor has been investigated as a modulator of apoptosis and has been shown to confer significant protective effects on cell death in neurodegenerative diseases. However, GSK-3β is known to have paradoxical effects on apoptosis subtypes, i.e., pro-apoptotic in mitochondrial-associated intrinsic apoptosis, but anti-apoptotic in death receptor-related extrinsic apoptosis. In this study, we evaluated the effect of a new GSK-3β inhibitor (JGK-263) on motor neuron cell survival and apoptosis, by using low to high doses of JGK-263 after 48 h of serum withdrawal, and monitoring changes in extrinsic apoptosis pathway components, including Fas, FasL, cleaved caspase-8, p38α, and the Fas–Daxx interaction. Cell survival peaked after treatment of serum-deprived cells with 50 μM JGK-263. The present study showed that treatment with JGK-263 reduced serum-deprivation-induced motor neuronal apoptosis by inactivating not only the intrinsic, but also the extrinsic apoptosis pathway. These results suggest that JGK-263 has a neuroprotective effect through effective modulation of the extrinsic apoptosis pathway in motor neuron degeneration.