Insulin Promotes Mitochondrial Respiration and Survival through PI3K/AKT/GSK3 Pathway in Human Embryonic Stem Cells.

Insulin is an essential growth factor for the survival and self-renewal of human embryonic stem cells (hESCs). Although it is best known as the principal hormone promoting glycolysis in somatic cells, insulin's roles in hESC energy metabolism remain unclear. In this report, we demonstrate that insulin is essential to sustain hESC mitochondrial respiration that is rapidly decreased upon insulin removal. Insulin-dependent mitochondrial respiration is stem cell specific, and mainly relies on pyruvate and glutamine, while glucose suppresses excessive oxidative phosphorylation. Pharmacologic and genetic manipulations reveal that continuous insulin signal sustains mitochondrial respiration through PI3K/AKT activation and downstream GSK3 inhibition. We further show that insulin acts through GSK3 inhibition to suppress caspase activation and rescue cell survival. This study uncovers a critical role of the AKT/GSK3 pathway in the regulation of mitochondrial respiration and cell survival, highlighting insulin as an essential factor for accurate assessment of mitochondrial respiration in hESCs.

Novel venom-based peptides (P13 and its derivative-M6) to maintain self-renewal of human embryonic stem cells by activating FGF and TGFß signaling pathways.

BACKGROUND: In our previous study, a venom-based peptide named Gonearrestide (also named P13) was identified and demonstrated with an effective inhibition in the proliferation of colon cancer cells. In this study, we explored if P13 and its potent mutant M6 could promote the proliferation of human embryonic stem cells and even maintain their self-renewal. METHODS: The structure-function relationship analysis on P13 and its potent mutant M6 were explored from the molecular mechanism of corresponding receptor activation by a series of inhibitor assay plus molecular and dynamics simulation studies. RESULTS: An interesting phenomenon is that P13 (and its potent mutant M6), an 18AA short peptide, can activate both FGF and TGFß signaling pathways. We demonstrated that the underlying molecular mechanisms of P13 and M6 could cooperate with proteoglycans to complete the "dimerization" of FGFR and TGFß receptors. CONCLUSIONS: Taken together, this study is the first research finding on a venom-based peptide that works on the FGF and TGF-ß signaling pathways to maintain the self-renewal of hESCs.

Ganoderma lucidum extract ameliorates MPTP-induced parkinsonism and protects dopaminergic neurons from oxidative stress via regulating mitochondrial function, autophagy, and apoptosis.

Neuroprotection targeting mitochondrial dysfunction has been proposed as an important therapeutic strategy for Parkinson's disease. Ganoderma lucidum (GL) has emerged as a novel agent that protects neurons from oxidative stress. However, the detailed mechanisms underlying GL-induced neuroprotection have not been documented. In this study, we investigated the neuroprotective effects of GL extract (GLE) and the underlying mechanisms in the classic MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced mouse model of PD. Mice were injected with MPTP to induce parkinsonism. Then the mice were administered GLE (400 mg kg-1 d-1, ig) for 4 weeks. We observed that GLE administration significantly improved locomotor performance and increased tyrosine hydroxylase expression in the substantia nigra pars compact (SNpc) of MPTP-treated mice. In in vitro study, treatment of neuroblastoma neuro-2a cells with 1-methyl-4-phenylpyridinium (MPP+, 1 mmol/L) caused mitochondrial membrane potential collapse, radical oxygen species accumulation, and ATP depletion. Application of GLE (800 µg/mL) protected neuroblastoma neuro-2a cells against MPP+ insult. Application of GLE also improved mitochondrial movement dysfunction in cultured primary mesencephalic neurons. In addition, GLE counteracted the decline in NIX (also called BNIP3L) expression and increase in the LC3-II/LC3-I ratio evoked by MPP+. Moreover, GLE reactivated MPP+-inhibited AMPK, mTOR, and ULK1. Similarly, GLE was sufficient to counteract MPP+-induced inhibition of PINK1 and Parkin expression. GLE suppressed MPP+-induced cytochrome C release and activation of caspase-3 and caspase-9. In summary, our results provide evidence that GLE ameliorates parkinsonism pathology via regulating mitochondrial function, autophagy, and apoptosis, which may involve the activation of both the AMPK/mTOR and PINK1/Parkin signaling pathway.

The Suppression of Medium Acidosis Improves the Maintenance and Differentiation of Human Pluripotent Stem Cells at High Density in Defined Cell Culture Medium.

Cell density has profound impacts on the cell culture practices of human pluripotent stem cells. The regulation of cell growth, cell death, pluripotency and differentiation converge at high density, but it is largely unknown how different regulatory mechanisms act at this stage. We use a chemically defined medium to systemically examine cellular activities and the impact of medium components in high-density culture. We show that medium acidosis is the main factor that alters cell cycle, gene expression and cellular metabolism at high cell density. The low medium pH leads to inhibition of glucose consumption, cell cycle arrest, and subsequent cell death. At high cell density, the suppression of medium acidosis with sodium bicarbonate (NaHCO3) significantly increases culture capacity for stem cell survival, derivation, maintenance and differentiation. Our study provides a simple and effective tool to improve stem cell maintenance and applications.

Neuroprotective effects of 5-(4-hydroxy-3-dimethoxybenzylidene)-thiazolidinone in MPTP induced Parkinsonism model in mice.

Parkinson's disease (PD) is a neurological disorder characterized by degeneration of nigrostriatal dopaminergic (DAergic) system. Present treatment targeting to DAergic system solely ameliorated the symptoms but failed to retard the DAergic neuron degeneration, therefore new therapeutic methods aiming at preventing or delaying the neurodegenerative process are urgently needed. In the present study, we found that 5-(4-hydroxy-3-dimethoxybenzylidene)-2-thioxo-4-thiazolidinone (RD-1), a compound derived from rhodanine, protected DAergicneurons from neurotoxicity of MPTP/MPP(+). Firstly, RD-1 significantly improved the locomotor ability in the MPTP mice model, and elevated the tyrosine hydroxylase (TH) positive cell numbers in substantianigra pars compacta (SNpc) and the integrated optical density (IOD) of TH-positive nerve fibers in striatum respectively. Since mitochondrial dysfunction plays an important role in pathogenesis of PD, thereby we investigated the molecular mechanisms of RD-1 against MPTP/MPP(+) neurotoxicity, focusing on its effects on the mitochondrial dysfunction. Immunoblotting analysis showed that RD-1 significantly elevated the Parkin and Miro2 expression levels in acute MPTP treated mice, and improved mitochondrial membrane potential and ATP synthesis in MPP(+)-treated Neuro-2a cells. Moreover, RD-1attenuated impaired mitochondrial transport and vesicle release dysfunction evoked by MPP(+) cytotoxicity in cultured primary mesencephalic neurons. Taken together, these results indicate that improving the mitochondrial dysfunction may be a good choice to delay the neurodegenerative progression commonly associated with PD.