Dual regulation of TxNIP by ChREBP and FoxO1 in liver

Abstract

TxNIP (Thioredoxin-interacting protein) is considered as a potential drug target for type 2 diabetes. Although TxNIP expression is correlated with hyperglycemia and glucotoxicity in pancreatic β cells, its regulation in liver cells has been less investigated. In the current study, we aim at providing a better understanding of Txnip regulation in hepatocytes in response to physiological stimuli and in the context of hyperglycemia in db/db mice. We focused on regulatory pathways governed by ChREBP (Carbohydrate Responsive Element Binding Protein) and FoxO1 (Forkhead box protein O1), transcription factors that play central roles in mediating the effects of glucose and fasting on gene expression, respectively. Studies using genetically modified mice reveal that hepatic TxNIP is up-regulated by both ChREBP and FoxO1 in liver cells and that its expression strongly correlates with fasting, suggesting a major role for this protein in the physiological adaptation to nutrient restriction.

Keywords: Endocrinology; Molecular Biology; Molecular Physiology.

Graphical abstract

Figure 1

TxNIP expression is increased in the liver of db/db mice Twelve-week-old C57BL/6J (+/+) and db/db male mice were fed ad libitum. Figures are presented as means ± SEM from 8 to 12 individual mice. Significance is based on two-way ANOVA followed by a Bonferroni post hoc test. ∗p < 0.5, ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0005 when compared with (+/+) mice. (A) Blood glucose (mM) recovered at the time of harvest from tail snip. (B) Relative Txnip gene expression determined by qPCR. (C) Western blot analysis of protein extracted from whole-liver lysate. HSP90 was used as loading control. Five representative samples are shown. (D) Quantification of the ratio of phosphorylated FoxO1 corrected to total FoxO1 protein, of the ratio of phosphorylated FoxO1 corrected to HSP90, and of total FoxO1 corrected to HSP90. (E) Relative Chrebpα, Chrebpβ, Lpk, Foxo1, Pepck, G6pase, and Igfbp1 gene expression determined by qPCR. (F) ChIP analysis followed by qPCR of whole mouse liver tissue. Immunoprecipitation experiments conducted with ChREBP antibodies. The DNA regions of the Lpk and Txnip promoters were amplified using primers indicated in Table S2.

Figure 2

Effect of ChREBP and FoxO1 overexpression on Txnip expression and promoter activity in mouse hepatocytes Primary hepatocytes derived from adult male mice were incubated under low glucose concentration (5 mM) with specific adenovirus as indicated (from 0.1 to 3.0 PFU/cell) for 24 h. (A) Relative Chrebp, Foxo1, Acc, Igfbp1, and Txnip gene expression determined by qPCR. Figures are presented as means ± SEM from 6 to 8 independent cultures. Significance is based on two-way ANOVA followed by a Bonferroni post hoc test †p < 0.05, ††p < 0.01, †††p < 0.001 when compared with GFP conditions. (B) Schematic representation of the wild and mutated Txnip promoters (1,081 bp; the two ChREBP-binding sites [ChoRE] and the FoxO1-binding site [IRE] are indicated). Primers used for site-directed mutagenesis are indicated in Table S1. (C) Luciferase activity of the wild-type Txnip promoter in primary hepatocytes in response to either ChREBP^CA (3 PFU/cell) or FoxO1^CA (3 PFU/cell). Figures are presented as means ± SEM from 6 to 8 independent cultures. Significance is based on two-way ANOVA followed by a Bonferroni post hoc test. ∗∗p < 0.01 when compared with GFP conditions. (D) Luciferase activity of the wild-type, ChoREa-mutated, or IRE-mutated Txnip promoter in primary hepatocytes in response to either ChREBP^CA (3 PFU/cell) or FoxO1^CA (3 PFU/cell). Figures are presented as means ± SEM from 5 to 8 independent cultures. Significance is based on two-way ANOVA followed by a Bonferroni post hoc test ∗p < 0.05, ∗∗∗p < 0.001 when compared with wild-type promoter activity.

Figure 3

Correlation between Txnip expression and blood glucose concentrations (A and B) Twelve-week-old C57BL/6J (+/+) male mice were studied at the fed, fasted, and refed state. (A) Western blot analysis of protein extracted from whole-liver lysate. HSP90 was used as loading control. Five representative samples are shown. (B) Relative Txnip gene expression determined by qPCR. Figure is presented as means ± SEM from 8 to 12 individual mice. Significance is based on two-way ANOVA followed by a Bonferroni post hoc test. ∗∗p < 0.01, ∗∗∗p < 0.001 when compared with the fasted state. (C–F) Representation of the relative amounts of Txnip mRNA levels as a function of glycemia. (C) Correlation using a set of C57BL/6J male mice (n = 32 points) measured at either the fasted, fed, and refed state. R, Spearman's correlation coefficient, ∗∗∗p < 0.001 (coefficient other than zero). Dashed oval, point cloud approximation. (D–F) Linear regressions for blood glucose concentrations below 6 mM (D, n = 11), between 6 and 13 mM (E, n = 15) and above 13 mM (F, n = 5). R2, coefficient R squared (adequacy of the points to the right); y = ax + b, equation of the regression line with a slope.

Figure 4

Differential regulation of TxNIP during fasting and refeeding. Adult C57BL/6J male mice were studied under fasting (24 h fast) or refed (a 18-h refeeding period) at ZT12 Data are expressed as means ± SEM, n = 6 to 8 individual mice/group. Significance is based on two-way ANOVA followed by a Bonferroni post hoc test ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 when compared with fasted conditions. (A) Blood glucose (mM) recovered at the time of harvest from tail snip. (B) Relative Txnip gene expression determined by qPCR. (C) Relative Chrebpα, Chrebpβ, Lpk, and Pepck, gene expression determined by qPCR. (D) Western blot analysis of protein extracted from whole-liver lysate. HSP90 was used as loading control. Four representative samples are shown. (E) Quantification of the ratio of phosphorylated FoxO1 corrected to total FoxO1 protein is provided. (F) ChIP analysis followed by qPCR of whole mouse liver tissue. Immunoprecipitation experiments conducted with FoxO1 and ChREBP antibodies. The DNA regions of the Pecpk, Lpk, and Txnip promoters were amplified using primers indicated in Table S1. Data are expressed as means ± SEM, n = 6 to 8 individual mice/group. Significance is based on two-way ANOVA followed by a Bonferroni post hoc test ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 when compared with fasted conditions.

Figure 5

The glucose-dependent induction of Txnip requires ChREBP C57Bl/6J male mice were injected intravenously with a single dose of 5 × 10⁹ PFU of shCTRL or shChREBP adenovirus at Day1. Seven days later, mice were challenged to nutritional manipulations as indicated (fasted or refed). (A) Western blot analysis of protein extracted from whole-liver lysate. HSP90 was used as loading control. Three representative samples are shown. Quantification of the ratio of TxNIP protein content corrected to HSP90 is shown. ∗p < 0.05 when compared with ShControl conditions. (B) Relative gene expression of Chrebpα, Chrebpβ, Lpk, Acc, and Txnip gene was determined by qPCR. Figures are presented as means ± SEM from 6 individual mice. Significance is based on two-way ANOVA followed by Bonferroni post test, ∗p < 0.05, ∗∗p < 0.01, when compared with refed ShControl conditions. (C) Primary hepatocytes from Chrebp^−/- and Chrebp^+/+ littermates were stimulated 1 day after platting for 24 h with cell culture medium containing 5 or 25 mM glucose. qPCR analysis of Chrebp, Chrebpβ, Lpk, Acc, and Txnip. Figures are presented as means ± SEM from 3 independent cultures done in triplicates. Significance is based on two-way ANOVA followed by Bonferroni post test, ∗p < 0.05, ∗∗∗p < 0.0 01.

Figure 6

Daily rhythms of hepatic gene expression under control, fasted, and refed conditions Adult C57BL/6J male mice were studied under control conditions (fed at libitum), fasted, or refed at ZT12. Mice were killed by cervical dislocation at several time points in a pairwise manner: ZT0, ZT4, ZT8, ZT12 ZT14, ZT16, ZT20, and ZT24 as indicated. Figures are presented as means ± SEM from 10 individual mice per time point. (A) Blood glucose (mmol/L) recovered at time of harvest from tail snip. (B) qPCR analysis of Reverbα. (C) qPCR analysis of Chrebpα, Chrebpβ, Lpk, Txnip, Pepck, and Foxo1.

Figure 7

FoxO1 stimulates the expression of TXNIP in liver (A and B) (A) Wild-type (WT) and LFoxO^TKO mice were fasted for 24 h qPCR analysis of Foxo1, Foxo3, Igfbp1, Txnip, Chrebpα, Chrebpβ, and Lpk. Data are expressed as means ± SEM, n = 6 to 8 individual mice/group. Significance is based on two-way ANOVA followed by Bonferroni post hoc test, ∗∗p < 0.01, ∗∗∗p < 0.001 when compared with WT mice. (B) Wild-type (WT), LIR^KO, and LIRFoxO1^KO mice were studied at the fed state. qPCR analysis of InsR, Txnip, Igfbp1, Pecpk, and Foxo1. Data are expressed as means ± SEM, n = 6 to 8 individual mice/group. Significance is based on two-way ANOVA followed by Bonferroni post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 when compared with LIR^KO mice. (C and D) Control (CTRL) and transgenic mice overexpressing a constitutive active form of FoxO1 (FoxO1^TGN) were studied at the fed state. (C) Western blot analysis of protein extracted from whole liver lysate. GAPDH was used as loading control. Four representative samples are shown. (D) qPCR analysis of Foxo1, Txnip, Pecpk Chrebpα, Chrebpβ, and Lpk. Data are expressed as means ± SEM, n = 6 individual mice/group. Significance is based on two-way ANOVA followed by Bonferroni post hoc test, Significance is based on two-way ANOVA followed by a Bonferroni post hoc test ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 8

Txnip silencing in liver reduces hyperglycemia in db/db mice Adult C57BL/6J (+/+) and db/db male mice were injected intravenously with a single dose of 5 × 10⁹ PFU of ShControl or ShTxnip adenovirus (GeneCust) at Day1. Seven days later, mice were challenged to a pyruvate tolerance test (PTT) or to nutritional manipulations as indicated (a 24-h fast [Fasted] or analyzed at the fed state [Fed]). Data are expressed as means ± SEM, n = 6 to 8 individual mice/group. Significance is based on two-way ANOVA followed by Bonferroni post hoc test, (A) Western blot analysis of protein extracted from whole-liver lysate. HSP90 was used as loading control. Four representative samples are shown. Quantification of the ratio of TxNIP protein to HSP90 is shown. ∗p<0.05 when compared to db/db ShControl. (B) Pyruvate tolerance test (PTT) †p < 0.05, ††p < 0.01, †††p < 0.001, ††††p < 0.0005 when compared with db/db ShControl.. (C) Blood glucose concentrations under fasted and fed state. (D) Body weight at the time of the sacrifice. (E) qPCR analysis of Txnip, Pecpk G6Pase, Foxo1, and PGC1α. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001 when compared to db/db ShControl.