3.2 GA I induces autophagy via inhibition of phosp...
We found that the number of autophagosome per cell，the ratio of LC3-II/LC3-I and the degradation of SQSTM1/p62 were increased after treatment with 5μg/mL GA I for 36 hr(Figure 2a-d).
Evaluating the number of cellular autophagosomes is generally but not always an indicator of the level of cellular autophagic activity.When the activity of lysosomal enzyme decreased，the autophagosomes will accumulate too，even under normal condition(Klionsky et al.，2016).To estimate the autophagic activity，we followed a classical experiment，LC3 Turnover Assay.Chloroquine，an autopha-gic inhibitor，inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal p H.GA I induced accumulation of LC3-Ⅱand the ratio of LC3-Ⅱis significantly increased by treatment with chloroquine plus GA I compared with chloroquine treated group，which indicated that autophagic flux was increased after GA I treatment(Figure 2g).
There is a classic pathway that regulates autophagy，the m TOR-dependent induced autophagy pathway.S6K is a substrate of mTOR and the serine70 phosphorylation activity is regulated by m TOR(Yoshii& Mizushima，2017).Our data showed that GA I inhibited the serine70 phosphorylation of S6K and serine 2448 phosphorylation of mTOR(Figure 2e，f).
Figure 1 GA I protects cells from high glucose induced apoptosis.(https://www.daowen.com)
(a)The structure of GA I.(b)MIN-6 cells were cultured with or without 5μg/mL GA I for 36 hr under the high glucose stress and the apoptotic rate of cells were tested by flow cytometry.Cells cultured with DMEM were used as control.DMEM：Dulbecco's modified Eagle's medium；GA I：gymnemic acid I［Color figure can be viewed at wileyonlinelibrary.com］
Figure 2 GA I induces autophagy in MIN-6 cells
(a)MIN-6 cells were treated with or without 5μg/mL of GA I for 36hr and stained with an anti-LC3 antibody(scale bar=5μm).(b)Cells were treated as(a)and the number of autophagosome per cells were counted.**p＜0.01，compared with the control group by Student's t test.At least 30 cells were counted.At least three replicates were performed.(c)-(e)The MIN-6 cells were treated with or without 5μg/mL of GA I.The cell extracts were immunoblotted with antibodies against LC3，SQSTM1/p62，S6K，or phospho-p70 S6K.GAPDH served as loading control.(f)The MIN-6 cells were treated with or without 5μg/mL of GA I.The cell extracts were immunoblotted with antibodies against mTOR or phospho-mTOR.(g)The MIN-6 cells were treated with or without 5μg/mL GA I，50μM chloroquine，5μg/mL GA I+50μM chloroquine and total cell extracts were immunoblotted with antibody against LC3 or GAPDH.GA I：gymnemic acid I；GAPDH：glyceraldehyde 3-phosphate dehydrogenase；LC：light chain；mTOR：mammalian target of rapamycin［Color figure can be viewed at wileyonlinelibrary.com］