Figure 5: TBP-2 suppresses glucose-induced mitochondrial energy production and insulin secretion in β-cells.

Transient TBP-2 knockdown by TBP-2 siRNA (RNAi1 and RNAi2) and negative control siRNA (NC) in INS-1 cells. The expression of TBP-2 was determined by qRT–PCR (a) at 48 h after transfection and by immunoblot analysis (b) at 72 h after transfection. (c) Transient TBP-2 overexpression in INS-1 cells after 24 h. The expression of TBP-2 was determined by immunoblot analyses. Augmentation or suppression of GSIS in TBP-2 knockdown (d) or TBP-2 overexpression (e) cells in RPMI cultured medium. Statistic insulin secretion assays were analysed in INS-1 cells. (f) Construction of the doxycycline (dox)-off-dependent TBP-2-overexpressed INS-1 cells. TBP-2 protein was suppressed by 1000 ng ml⁻¹ dox and induced by dox removal. Cells were cultured with (+, open bar) or without (−, closed bar) dox for 24 h. INS-1 cells were incubated at low (2.8 or 3 mM) or high (16.7 or 20 mM) glucose and experiments were performed. Suppression of high (20 mM) GSIS (g), high (16.7 mM) glucose-enhanced ATP contents (h) and intracellular Ca²⁺ influx (i) by dox-off TBP-2 overexpression, but not KCl-induced insulin secretion (g). The inset bar graph shows area under the curve levels (%) of intracellular Ca²⁺ levels between 320 and 900 s (i). Decrease of mitochondrial membrane potentials (MMP) in dox-off TBP-2 overexpression in INS-1 cells. MMP of cultured cells in medium containing 3 mM (upper panel) or 20 mM (lower panel) glucose for 24 h was analysed by flow cytometry (j) or in cultured medium by fluorescence microscopy (k) using jc-1 reagent, scale bar is 100 μm. For disruption of MMP, 5 μM carbonyl cyanide m-chlorophenlhydrazone (cccp) reagent was used. Flow cytometer, blue; dox (+), red; dox (−), black; dox (+) + cccp, grey; dox (−) + cccp. (l) Glucokinase (GK) activities in INS-1 cells, NS, nonsignificant. (m) Suppression of pyruvate (sodium pyruvate) or 2-ketoisohexanoic acid (KIC) and methylsaccinic acid (monomethyl succinate)-stimulated insulin secretion by dox-off-dependent TBP-2 overexpression. (n) A flow cytometric analyses with Annexin V-fluorescein isothiocyanate and propidium iodide staining in the dox-off TBP-2-overexpressed INS-1 cells. Preapoptotic cells were calculated from triplicate samples (%). Data are presented as mean±s.d. *P<0.05, **P<0.01, ***P<0.001, versus control (t-test).