Thioredoxin-interacting protein (Txnip) gene expression: sensing oxidative phosphorylation status and glycolytic rate

Abstract

Thioredoxin-interacting protein (Txnip) has important functions in regulating cellular metabolism including glucose utilization; the expression of the Txnip gene is sensitive to the availability of glucose and other fuels. Here, we show that Txnip expression is down-regulated at the transcriptional level by diverse inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). The effect of these OXPHOS inhibitors is mediated by earlier identified carbohydrate-response elements (ChoREs) on the Txnip promoter and the ChoRE-associated transcription factors Max-like protein X (MLX) and MondoA (or carbohydrate-response element-binding protein (ChREBP)) involved in glucose-induced Txnip expression, suggesting that inhibited oxidative phosphorylation compromises glucose-induced effects on Txnip expression. We also show that the OXPHOS inhibitors repress the Txnip transcription most likely by inducing the glycolytic rate, and increased glycolytic flux decreases the levels of glycolytic intermediates important for the function of MLX and MondoA (or ChREBP). Our findings suggest that the Txnip expression is tightly correlated with glycolytic flux, which is regulated by oxidative phosphorylation status. The identified link between the Txnip expression and glycolytic activity implies a mechanism by which the cellular glucose uptake/homeostasis is regulated in response to various metabolic cues, oxidative phosphorylation status, and other physiological signals, and this may facilitate our efforts toward understanding metabolism in normal or cancer cells.

FIGURE 1.

OXPHOS inhibitors compromise Txnip mRNA expression. HeLa cells were treated with the indicated OXPHOS inhibitors for 0, 1, 2, and 4 h, and the Txnip mRNA expression levels were scored by quantitative real-time PCR. The dosages of the drugs used were within reported effective ranges, which are: rotenone, 1 μm; 2-thenoyltrifluoroacetone (TTFA), 2 μm; antimycin A, 1 μg/ml; myxothiazol, 1 μm; NaN3, 1 mm; DETA/NO, 1 mm; oligomycin A, 1 μg/ml; and carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 1 μm. The same dosages were used throughout this study. H2B, histone 2B. Error bars indicate S.E.

FIGURE 2.

Decreased protein expression levels in cells treated by OXPHOS inhibitors. A, HeLa cells stably expressing a transgenic cytomegalovirus-driven GFP-Txnip fusion gene were treated with 40 μm cycloheximide (CHX) for the indicated times followed by immunoblot analysis of whole cell lysates. B, HEK293 cells were treated with 40 μm cycloheximide for the indicated times or with 20 μm proteasome inhibitor MG-132 for 12 h followed by analysis of endogenous Txnip expression. C and D, HeLa cells (C) and HEK293 cells (D) were treated with mitochondrial inhibitors for the indicated times followed by immunoblot analysis of whole cell lysates.

FIGURE 3.

Repressed Txnip promoter activity in cells treated with NaN₃ or rotenone. A, a diagram shows the luciferase reporter constructs used in B; Mut-ChoRE contains mutations at two ChoRE sites, and WT-Short is a hybrid promoter that contains a shorter Txnip promoter segment and the TATA box in front of the luciferase gene. WT, wild type. B, luciferase activities of the indicated reporter genes in HeLa cells treated with NaN₃ or rotenone, expressed as the percentages of the corresponding activities in untreated cells. Error bars indicate S.E.

FIGURE 4.

The occupancy of Mondo-MLX on the Txnip promoter is reduced by NaN₃ or rotenone. A, the protein level of ectopic GFP-MondoA and FLAG-MLX or endogenous ChREBP was not affected by NaN₃ or rotenone treatment (1.5 h) in HeLa or INS-1 cells, whereas Txnip expression was repressed. B and C, ChIP assays in HeLa cells, respectively, treated (1 h) with NaN₃ and rotenone, using antibodies corresponding to the indicated proteins. NF-YA, nuclear factor Y, subunit A; Pol II, RNA polymerase II. D, ChIP assay in pancreatic INS-1 cells treated with NaN₃ (1 h) using antibodies corresponding to the indicated proteins. The occupancy was scored by quantitative real-time PCR, expressed as percentages of input total chromatin DNA. Error bars in B and D indicate S.E.

FIGURE 5.

OXPHOS inhibitors repress glucose-induced Txnip expression. HeLa cells were first incubated in glucose-free medium overnight and were left untreated or treated with 10 mm glucose for 2 h (A) or 10 mm 2DG for 1 h (B) with or without OXPHOS inhibitors. Txnip mRNA levels were scored by real-time PCR. Error bars indicate S.E.

FIGURE 6.

AMPK does not affect the Txnip expression patterns. A, control HeLa cells or cells preincubated (20 min) with 10 μm compound C were treated with NaN₃ or rotenone (1 h) or left untreated, and the Txnip mRNA levels were scored by real-time PCR. B, the AMPKα protein level in HeLa cells transfected with a mixture of siRNAs targeting AMPKα1 and AMPKα2 was significantly reduced, as indicated by immunoblot analysis of whole cell lysates (left; please note that anti-AMPKα antibodies detect both isoforms). The effect of rotenone (Rot) on Txnip mRNA expression was not reversed by AMPKα siRNAs either singly or in combination (right). Error bars indicate S.E.

FIGURE 7.

Txnip transcription can sense glycolytic rate. A, HeLa cells were left untreated or treated with indicated OXPHOS inhibitors for 1, 2, or 4 h, and lactate levels in the medium were determined. B, HeLa cells maintained in glucose-containing medium were treated with the indicated glycolytic intermediates for 2 h (G6P, 25 mm; GADP, 5 mm; 3-phosphoglycerate (3PG) 5 mm) with or without rotenone. C, HeLa cells, preincubated with 0.5 mm iodoacetate (IodoAc), 0.5 mm sodium acetate (NaAC), or 10 mm oxamate for 20 min, were incubated in the absence or presence of NaN₃ or rotenone for 2 h. D, the GAPDH protein level in HeLa cells transfected with two siRNAs targeting GAPDH was significantly reduced, as indicated by immunoblot analysis of whole cell lysates (left). The effect of rotenone on Txnip expression was reversed by GAPDH-specific siRNAs (right). In B–D, Txnip mRNA levels were scored by real-time PCR. Error bars in B–D indicate S.E.

FIGURE 8.

A model linking Txnip expression, glucose transport, glycolysis, OXPHOS, and other physiological cues. OXPHOS inhibitors (this work), glutamine (22), and insulin signaling (9) have all been shown to repress Txnip expression; these factors are also known to increase the glycolytic rate. The augmented glycolytic flux may dynamically deplete glycolytic intermediate metabolites normally involved in transmitting glucose signaling to Mondo-MLX activation, which in turn represses Txnip expression. Our data suggest that the metabolites upstream of the GAPDH enzyme, G6P and GADP in particular but others are not ruled out, are candidates that link Txnip expression to glycolytic flux. How Mondo-MLX transcription factors are activated, the nuclear translocation included, and whether the above mentioned relevant metabolites are directly involved in this activation process are a challenging problem to be addressed in the future. Txnip has an inhibitory role on glucose transport; hence, glucose uptake in cells with repressed Txnip expression will be induced, a common theme in many cancerous cells that are addictive to glucose. Glut, glucose transporters; 3PG, 3-phosphoglycerate.