Metformin induces mitochondrial remodeling and differentiation of pancreatic progenitor cells into beta-cells by a potential mechanism including suppression of the T1R3, PLCß2, cytoplasmic Ca+2, and AKT.

The main goal of this study was to investigate the molecular changes in pancreatic progenitor cells subject to high glucose, aspartame, and metformin in vitro. This scope of work glucose, aspartame, and metformin were exposed to pancreatic islet derived progenitor cells (PID-PCs) for 10 days. GLUT1's role in beta-cell differentiation was examined by using GLUT1 inhibitor WZB117. Insulin+ cell ratio was measured by flow cytometry; the expression of beta-cell differentiation related genes was shown by RT-PCR; mitochondrial mass, mitochondrial ROS level, cytoplasmic Ca2+, glucose uptake, and metabolite analysis were made fluorometrically and spectrophotometrically; and proteins involved in related molecular pathways were determined by western blotting. Findings showed that glucose or aspartame exposed cells had similar metabolic and gene expression profile to control PID-PCs. Furthermore, relatively few insulin+ cells in aspartame treated cells were determined. Aspartame signal is transmitted through PLCß2, CAMKK2 and LKB1 in PID-PCs. The most obvious finding of this study is that metformin significantly increased beta-cell differentiation. The mechanism involves suppression of the sweet taste signal's molecules T1R3, PLCß2, cytoplasmic Ca+2, and AKT in addition to the direct effect of metformin on mitochondria and AMPK, and the energy metabolism of PID-PCs is remodelled in the direction of oxidative phosphorylation. These findings are very important in terms of determining that metformin stimulates the mitochondrial remodeling and the differentiation of PID-PCs to beta-cells and thus it may contribute to the compensation step, which is the first stage of diabetes development.

Phospholipase C inhibits apoptosis of porcine oocytes cultured in vitro.

Oocyte apoptosis can be used as an indicator of oocyte quality and development competency. Phospholipase C (PLC) is a critical enzyme that participates in phosphoinositide metabolic regulation and performs many functions, including the regulation of reproduction. In this study, we aimed to explore whether PLC participates in the regulation of apoptosis in porcine oocytes and investigated its possible mechanism. In porcine oocytes, 0.5 µM U73122 (the PLC inhibitor) was considered to be the best concentration to facilitate maturation, and 0.5 µM m-3M3FBS (the PLC activator) was regarded as the most appropriate concentration to inhibit maturation. The percentage of cleavage and blastocysts treated with 0.5 µM U73122 was lower than that of the control group. Furthermore, the percentage of cleavage and blastocysts treated with 0.5 µM m-3M3FBS was higher than that of the control group. The relative PLC messenger RNA (mRNA) expression tested by a quantitative real-time polymerase chain reaction was found to be inhibited by 0.5 µM U73122 or activated by 0.5 µM m-3M3FBS. The relative mRNA abundance of BAK, BAX, CASP3, CASP8, and TP53 and protein abundance of Bak, cleaved caspase-3, caspase-8, and P53 was activated by U73122 or inhibited by m-3M3FBS, while the relative mRNA and protein level of BCL6 showed the opposite trend. The intracellular Ca2+ concentration increased and the expression of PLCB1 protein also increased in porcine oocytes when they were cultured with 0.5 µM m-3M3FBS for 44 hours. The abundance of proteins PKCß and CAMKIIα and the expression of several downstream genes (CDC42, NFATc1, NFATc2, NFκB, and NLK) were activated by m-3M3FBS or inhibited by U73122. Our findings indicate that PLC inhibits apoptosis and maturation in porcine oocytes. The intracellular Ca2+ concentration, two Ca2+ -sensitive proteins, and several downstream genes were positively regulated by PLC.

The N-terminal homology (ENTH) domain of Epsin 1 is a sensitive reporter of physiological PI(4,5)P2 dynamics.

Phospholipase Cß (PLCß)-induced depletion of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) transduces a plethora of signals into cellular responses. Importance and diversity of PI(4,5)P2-dependent processes led to strong need for biosensors of physiological PI(4,5)P2 dynamics applicable in live-cell experiments. Membrane PI(4,5)P2 can be monitored with fluorescently-labelled phosphoinositide (PI) binding domains that associate to the membrane depending on PI(4,5)P2 levels. The pleckstrin homology domain of PLCδ1 (PLCδ1-PH) and the C-terminus of tubby protein (tubbyCT) are two such sensors widely used to study PI(4,5)P2 signaling. However, certain limitations apply to both: PLCδ1-PH binds cytoplasmic inositol-1,4,5-trisphosphate (IP3) produced from PI(4,5)P2 through PLCß, and tubbyCT responses do not faithfully report on PLCß-dependent PI(4,5)P2 dynamics. In searching for an improved biosensor, we fused N-terminal homology domain of Epsin1 (ENTH) to GFP and examined use of this construct as genetically-encoded biosensor for PI(4,5)P2 dynamics in living cells. We utilized recombinant tools to manipulate PI or Gq protein-coupled receptors (GqPCR) to stimulate PLCß signaling and characterized PI binding properties of ENTH-GFP with total internal reflection (TIRF) and confocal microscopy. ENTH-GFP specifically recognized membrane PI(4,5)P2 without interacting with IP3, as demonstrated by dialysis of cells with the messenger through a patch pipette. Utilizing Ci-VSP to titrate PI(4,5)P2 levels, we found that ENTH-GFP had low PI(4,5)P2 affinity. Accordingly, ENTH-GFP was highly sensitive to PLCß-dependent PI(4,5)P2 depletion, and in contrast to PLCδ1-PH, overexpression of ENTH-GFP did not attenuate GqPCR signaling. Taken together, ENTH-GFP detects minute changes of PI(4,5)P2 levels and provides an important complementation of experimentally useful reporters of PI(4,5)P2 dynamics in physiological pathways.

Supraspinal Gbetagamma-dependent stimulation of PLCbeta originating from G inhibitory protein-mu opioid receptor-coupling is necessary for morphine induced acute hyperalgesia.

Although alterations in micro-opioid receptor (microOR) signaling mediate excitatory effects of opiates in opioid tolerance, the molecular mechanism for the excitatory effect of acute low dose morphine, as it relates to microOR coupling, is presently unknown. A pronounced coupling of microOR to the alpha subunit of G inhibitory protein emerged in periaqueductal gray (PAG) from mice systemically administered with morphine at a dose producing acute thermal hyperalgesia. This coupling was abolished in presence of the selective microOR antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) administered at the PAG site, showing that the low dose morphine effect is triggered by microOR activated G inhibitory protein at supraspinal level. When Gbetagamma downstream signalling was blocked by intra-PAG co-administration of 2-(3,4,5-trihydroxy-6-oxoxanthen-9-yl)cyclohexane-1-carboxylic acid, a compound that inhibits Gbetagamma dimer-dependent signaling, a complete prevention of low dose morphine induced acute thermal hyperalgesia was obtained. Phospholipase C beta3, an enzyme necessary to morphine hyperalgesia, was revealed to be associated with Gbetagamma in PAG. Although opioid administration induces a shift in microOR-G protein coupling from Gi to Gs after chronic administration, our data support that this condition is not realized in acute treatment providing evidence that a separate molecular mechanism underlies morphine induced acute excitatory effect.

A novel Gbetagamma-subunit inhibitor selectively modulates mu-opioid-dependent antinociception and attenuates acute morphine-induced antinociceptive tolerance and dependence.

The Gbetagamma subunit has been implicated in many downstream signaling events associated with opioids. We previously demonstrated that a small molecule inhibitor of Gbetagamma-subunit-dependent phospholipase (PLC) activation potentiated morphine-induced analgesia (Bonacci et al., 2006). Here, we demonstrate that this inhibitor, M119 (cyclohexanecarboxylic acid [2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-(9Cl)]), is selective for mu-opioid receptor-dependent analgesia and has additional efficacy in mouse models of acute tolerance and dependence. When administered by an intracerebroventricular injection in mice, M119 caused 10-fold and sevenfold increases in the potencies of morphine and the mu-selective peptide, DAMGO, respectively. M119 had little or no effect on analgesia induced by the kappa agonist U50,488 or delta agonists DPDPE or Deltorphin II. Similar results were obtained in vitro, as only activation of the mu-opioid receptor stimulated PLC activation, whereas no effect was seen with the kappa- and delta-opioid receptors. M119 inhibited mu-receptor-dependent PLC activation. In studies to further explore the in vivo efficacy of M119, systemic administration M119 also resulted in a fourfold shift increase in potency of systemically administered morphine. Of particular interest, M119 was also able to attenuate acute, antinociceptive tolerance and dependence in mice treated concomitantly with both M119 and morphine. These studies suggest that small organic molecules, such as M119, that specifically regulate Gbetagamma subunit signaling may have important therapeutic applications in enhancing opioid analgesia, while attenuating the development of tolerance and dependence.