Cell experiment:

105 HepG2 cells are seeded in 12 well plates and cultured for 24 h in EMEM containing 10 % FCS. Cells are then serum starved in EMEM for 24 h and stimulated for 12 h with 10 nM insulin in the presence of SecinH3, the negative control D5 or vehicle (0.2% final concentration of DMSO). Total mRNA is prepared using Kit and cDNA for qPCR is generated from 1 μg RNA. qPCR is performed and data are normalized to β2-microglobulin expression[1].

Animal experiment:

C57/Bl6N mice are kept on a 12 h light/dark cycle in a pathogen-free animal facility and fed ad libitum with standard mice diet. After feeding with standard diet or with the same diet containing 0.9 μmol/g SecinH3 for 3 days, mice are intraperitoneally injected with 100 μL saline containing or not 40 μg recombinant human insulin. After 10 min the mice are anaesthetized and the liver is removed and lysed in lysis buffer. Normalized amounts of protein are either separated by SDS-PAGE and transferred onto nitrocellulose, or immunoprecipitated using agarose-conjugated antibodies against IRβ or IRS1[1].

SecinH3 is an antagonist of cytohesins with IC50s of 5.4 μM, 2.4 μM, 5.4 μM, 5.6 μM, 5.6 μM and 65 μM for hCyh1, hCyh2, mCyh3, hCyh3, drosophila steppke and yGea2-S7, respectively.
SecinH3 is a Sec7-specific guanine nucleotide exchange factor (GEF) inhibitor with preference for the small GEFs of the cytohesin family. SecinH3 almost completely blocks the insulin-dependent transcriptional repression of IGFBP1 with an IC50 of 2.2 μM. Insulin-stimulated translocation of ARF6 to the plasma membrane is also inhibited by SecinH3. It is found that SecinH3 inhibits the insulin-dependent phosphorylation of Akt and FoxO1A in a concentration-dependent manner. Insulin-induced exclusion of FoxO1A from the nucleus is completely prevented by SecinH3. The binding of IRS1 to the insulin receptor is also inhibited by SecinH3[1].
Compare to mice fed the same chow without SecinH3, the expression levels of the insulin-repressed gluconeogenic genes are elevated, whereas the insulin-induced glycolytic genes are reduced in SecinH3-treated mice. Insulin-stimulated Akt phosphorylation is also inhibited in SecinH3-treated mice. The expression of the genes for two key enzymes of mitochondrial β-oxidation, carnitine palmitoyltransferase 1a (Cpt1a) and hydroxyacyl-CoA dehydrogenase (Hadha), both of which are repressed by insulin, is increased in the SecinH3-treated mice. It is found significantly increased levels of serum insulin with slightly elevated glucose concentrations in SecinH3-treated mice. Accordingly, 3-hydoxybutyrate is increased in the serum of SecinH3-treated mice[1].
Reference:
[1]. Hafner M, et al. Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance. Nature. 2006 Dec 14;444(7121):941-4.