Ived either EGF (five ng/mL) or FCS 10 at day five or either LIF or CNTF (5 ng/mL) at DIV four and 6 ahead of total RNA extraction and quantitative real-time RT-PCR evaluation. Information are imply .e.m. (n = three) for every situation. Statistical analysis was performed making use of ANOVA followed by Dunnett’s test. P 0.01 versus EGF treatment; P 0.001 versus EGF therapy. A single representative experiment shown repeated thrice with related outcomes.contrast, CNTF only induced Ubiquitin Conjugating Enzyme E2 G2 Proteins Species glycogen synthase mRNA expression.DiscussionIn a prior study, it was shown that EGF maintains stem cells in an undifferentiated state, enhancing nestin Ubiquitin-Specific Peptidase 44 Proteins site expression and preventing them from spontaneously expressing characteristic markers of astrocytes (for example GFAP) or displaying metabolic characteristics (such as glutamate uptake) (Brunet et al, 2004). Accordingly, glycogen levels discovered in neural stem cells treated with EGF have been barely detectable, indicating that glycogen metabolism is not connected with such an undifferentiated stage. Exposure to FCS is actually a classic mean to receive differentiated astrocytes from neural stem cells. Fetal calf serum was found to induce the expression of glutamine synthetase (Loo et al, 1995), an enzyme typically linked with mature astrocytes as it participates to glutamate recycling, which can be a significant astrocytic function (Erecinska and Silver, 1990). Our preceding data also showed dramatic effects of FCS on quite a few distinct astroglial proteins and/or mRNAs which include GFAP, vimentin, or S100b, and on expression of essential metabolic proteins such as the glutamate aspartate transporter (GLAST), the monocarboxylate transporter 1 (MCT1), plus the a2-subunit in the Na + /K + ATPase (Brunet et al, 2004). In addition, metabolic characteristics of mature astrocytes like glutamate uptake or glutamate-induced activation of glycolysis emerged just after remedy with FCS. Glycogen metabolism also seems to become linked with maturation of astrocytes. Thus, the cellular glycogen contentJournal of Cerebral Blood Flow Metabolism (2010) 30, 51increased drastically immediately after FCS exposure. Cells also responded to forskolin treatment by exhibiting each a short-term glycogenolysis plus a long-term, overcompensated glycogen resynthesis, two phenomena previously described both in vitro and in vivo (Sorg and Magistretti, 1991, 1992; Swanson et al, 1992; Oz et al, 2009). In parallel, FCS-treated cells had enhanced mRNA expression of 3 important proteins involved in glycogen metabolism, namely glycogen synthase, glycogen phosphorylase, and PTG. The observation regarding PTG is specifically interesting as this protein was identified to become crucial for the regulation of glycogen metabolism in astrocytes (Allaman et al, 2000). On this basis, it can be proposed that PTG expression could be a important marker to identify mature astrocytes both in vitro and in vivo (Lovatt et al, 2007). Distinct growth variables happen to be identified as important components in gliogenesis. Amongst them, the family of interleukin-6 sort cytokines which consists of CNTF and LIF, occupies a central function (Lee et al, 2000) Various reports have recommended that CNTF and LIF can induce the differentiation of stem cells isolated at distinct embryonic ages into astrocytes, as determined by the expression of GFAP (Rajan and McKay, 1998). Indeed, both CNTF and LIF had been identified to boost GFAP expression in our stem cell cultures. Interestingly, the influence of each aspect on glycogen metabolism was distinct. Ciliary Neurotrophic Aspect modestly enhanced glycogen levels, wh.
