Regulation of TXNIP mRNA levels in β-cells.

<p>Transcription of TXNIP is under the control of a protein complex consisting of ChREBP (carbohydrate-responsive element-binding protein) and MondoA as well as the histone acetyltransferase P300. ChREBP is negatively regulated by AMPK. Consequently, stimulation of AMPK by AICAR inhibits ChREBP and TXNIP expression, while inhibition of AMPK by glucose and BML-275 activates ChREBP and increases TXNIP mRNA levels. Histone deacetylase 1 (HDAC1), counteracts P300-mediated histone acetylation, and is involved in insulin-mediated downregulation of TXNIP expression. Thus, inhibition of PI3K, AKT or HDAC1/3 increases TXNIP mRNA levels. Fatty acids-mediated stimulation of insulin secretion occurs via FFAR1/GPR40, a signalling pathway not involved in regulation of TXNIP expression. Fatty acids activate AMPK and have an additional effect on TXNIP mRNA levels. Fatty acids counteract glucose-induced TXNIP expression and ROS elevation, events which do not impede the ER strees-associated lipotoxic effect.</p

Inhibition of histone deacetylases antagonizes the time-dependent decline of TXNIP without reversing the effect of palmitate.

<p>INS-1E cells were cultured in the presence of 11 mM glucose and test substances as indicated and described under Materials and methods. (A,D,F) Changes of TXNIP mRNA levels (ΔΔCt) vs control set to 100% in A and D; vs max effect in F. (B,E,G) Representative western blots of ¹⁷²Thr-P-AMPKα and AMPKα1; Tubulin is used as loading control. (C) Quantitative analysis of western blots of three independent experiments of B. Results are given as mean ± SEM of n = 3–4 independent experiments. *p<0.05, **p<0.01, *** p<0.001 denotes significance to control, i.e. 11 mM Glc at 1h; ^<b>##</b>p<0.01 to 11 mM Glc at 24 h. $$p<0.01 significant effect of palmitate in the presence of MS. §§p<0.01 significant effect of palmitate in the presence of MS and BML. Abbreviations: Glc, glucose; Pal, palmitate; MS, MS-275 (HDAC1/2/3 inhibitor); BML, BML-275 (AMPK inhibitor); AICAR, AMPK activator.</p

Palmitate-mediated augmentation of GSIS occurs independently of TXNIP mRNA regulation.

<p>INS-1E cells were incubated for 1 h in KRB supplemented with test substances as described under Materials and methods. (A and B) Parallel measurement of (A) insulin secretion and (B) changes of TXNIP mRNA levels (ΔΔCt). (C) Insulin secretion of INS-1E cells after transient transfection with TXNIP or control virus. Data are expressed as mean ± SEM of n = 3–4 independent experiments. **p<0.01 denotes significance to the respective 2.8 mM Glc; ^<b>#</b>p<0.05, ^<b>##</b>p<0.01 to 12 mM Glc; ^<b>$$</b>p<0.01 to 20 mM Glc; ^<b>§§</b>p<0.001 to control virus transfected cells at 20 mM Glc; ^<b>&&</b>p<0.01 significant effect of palmitate vs 20 mM Glc in TXNIP overexpressing cells. Abbreviations: Glc, Glucose, Pal, palmitate.</p

The time-dependent reduction of TXNIP mRNA levels correlates with insulin/IGF-1 receptor activation.

<p>(A, C-F) INS-1E cells and (B) isolated mouse islets were cultured in the presence of test substances as indicated and described under Materials and methods. (A and B) Correlation of insulin released into the medium with TXNIP mRNA levels of (A) INS-1E cells and (B) mouse islets; (C) Changes of TXNIP mRNA levels (ΔΔCt) vs control (11 mM glucose at 24 h) set to 100%; (D-F) The IR/IGF1R inhibitor linsitinib was applied for 2 h, insulin/IGF-1 the second hour of incubation; (D) Relative <i>Txnip</i> mRNA levels (ΔCt) to the house keeping gene (<i>Rps13</i>). (E) Representative western blot of TXNIP and (F) quantitative analysis of three independent experiments; GAPDH was used as loading control. Results are presented as mean ± SEM of n = 3–4 independent experiments. *p<0.05, **p<0.01 significant vs 2.8 mM Glc; $p<0.05 significant vs 11 mM Glc; ^<b>#</b>p<0.05, ^<b>##</b>p<0.01 significant effect of linsitinib (“, unpaired t-test). Abbreviations: Glc, glucose; Ins, insulin; LY29, LY294002 (PI3K inhibitor); Akti, AKT/PKB inhibitor.</p

Palmitate- and time-dependent reduction of TXNIP expression despite stimulatory concentrations of glucose.

<p>INS-1E cells were cultured in the presence of test substances as indicated and described under Materials and methods. (A, E) Relative T<i>xnip</i> mRNA levels (ΔCt) to the house keeping gene (<i>Rps13</i>); (B, F) representative western blots; (C, G) quantitative analysis of western blots of three independent experiments presented as mean ± SEM; (D) Representative pictures of immunostained INS-1E cells for TXNIP (green) and nuclei (red). **p<0.01, ***p<0.001 vs 2.8 mM Glc, 1h; ^#p<0.05, ^##p<0.01, ^### p<0.001 significant effect of palmitate to the respective Glc concentration at the same time point; ^$$p<0.01 significant vs 11 mM Glc, 1h; Abbreviations: Glc, glucose; Pal, palmitate.</p

FFAR1/GPR40-independent effect of palmitate on TXNIP mRNA levels in human and mouse islets and INS-1E cells.

<p>(A and D) Isolated human islets, (B and E) mouse islets and (C) INS-1E cells were cultured for 24 h in the presence of test substances as indicated and described under Materials and methods. Changes in TXNIP mRNA levels (ΔΔCt) vs respective control (set to 100%, first column of each graph) are presented as mean ± SEM of n = 3–4 independent experiments; *p<0.05, **p<0.01, ***p<0.001 significant vs respective control. Abbreviations: Glc, glucose; Pal, palmitate; Ole, oleate; Stea, stearate; TUG, TUG-469 (FFAR1 agonist).</p