Glucose sensing by MondoA:Mlx complexes: a role for hexokinases and direct regulation of thioredoxin-interacting protein expression

Abstract

Glucose is a fundamental metabolite, yet how cells sense and respond to changes in extracellular glucose concentration is not completely understood. We recently reported that the MondoA:Mlx dimeric transcription factor directly regulates glycolysis. In this article, we consider whether MondoA:Mlx complexes have a broader role in sensing and responding to glucose status. In their latent state, MondoA:Mlx complexes localize to the outer mitochondrial membrane, yet shuttle between the mitochondria and the nucleus. We show that MondoA:Mlx complexes accumulate in the nucleus in response to glucose and 2-deoxyglucose (2-DG). Furthermore, nuclear localization of MondoA:Mlx depends on the enzymatic activity of hexokinases. These enzymes catalyze conversion of glucose to glucose-6-phosphate (G6P), which is the first step in the glycolytic pathway. Together, these findings suggest that MondoA:Mlx monitors intracellular G6P concentration and translocates to the nucleus when levels of this key metabolite increase. Transcriptional profiling experiments demonstrate that MondoA is required for >75% of the 2-DG-induced transcription signature. We identify thioredoxin-interacting protein (TXNIP) as a direct and glucose-regulated MondoA:Mlx transcriptional target. Furthermore, MondoA:Mlx complexes, via their regulation of TXNIP, are potent negative regulators of glucose uptake. These studies suggest a key role for MondoA:Mlx complexes in the adaptive transcriptional response to changes in extracellular glucose concentration and peripheral glucose uptake.

Fig. 1.

MondoA is a glucose sensor. (A) Rat L6 cells expressing MondoA and Mlx were starved overnight and treated with glucose or 2-DG as indicated. Subcellular localization of MondoA was determined after 6 h of treatment. Localization of Mlx was similar (data not shown). (B) Representative images of MondoA subcellular localization in the absence or presence of 2-DG. (Magnification: ×40.)

Fig. 2.

Nuclear translocation and accumulation of MondoA is an immediate-early response to 2-DG and requires Mlx. (A) L6 cells were treated with 2-DG in the presence or absence of cycloheximide, and the amount of nuclear MondoA was determined at the indicated time points. (B) L6 cells expressing MondoA alone or MondoA and Mlx as indicated were treated with 2-DG, and the subcellular localization of MondoA was determined after 3 h. (C and D) L6 cells expressing WT MondoA or the dimerization-defective MondoA mutant, MondoA(I766P), in combination with Mlx were treated with 2-DG and the nuclear localization of MondoA or MondoA(I766P) (C) or Mlx in the presence of MondoA(I766P) (D), determined after 3 h.

Fig. 3.

Nuclear translocation of MondoA depends on hexokinase activity. (A) HA1ER cells expressing MondoA and Mlx were treated with the indicated sugars, and subcellular localization of MondoA was determined after 3 h. (B) HA1ER cells expressing MondoA and Mlx were treated with 2-DG in the presence of the indicated increasing concentrations of 3-BrPA, and subcellular localization of MondoA was determined after 3 h. (C) HEK293T cells transfected with WT HKII or a catalytically impaired HKII mutant, HKII(D657A), were treated with 2-DG, and the subcellular localization of MondoA was determined after 3 h. (D) Expression of HKII and HKII (D657A) was identical in this experiment.

Fig. 4.

MondoA is a master regulator of glucose-induced transcription. Genes induced >1.8-fold (P < 0.005) after a 3-h 2-DG treatment in control and MondoA knockdown cells were determined by using Agilent 44K human microarrays. The 10 most highly regulated genes in each class are indicated along with their fold up-regulation in the two cell populations. (Inset) MondoA levels in the control and knockdown cells used for this experiment.

Fig. 5.

MondoA is a direct and glucose-dependent regulator of TXNIP. (A) qRT-PCR was used to determine the relative expression of TXNIP in HA1ER cells treated as indicated. (B) Expression of TXNIP, MondoA, and tubulin was determined by Western blotting in control and MondoA knockdown HA1ER cells. Cells were treated as indicated. (C) ChIP was used to determine MondoA occupancy at the TXNIP promoter in HA1ER cells under the indicated conditions.

Fig. 6.

MondoA is a negative regulator of glucose uptake. (A) TXNIP and MondoA expression in HA1E cells expressing ΔN237NLSMondoA (ΔN237), or vector alone, was determined by Western blotting. (B) Glucose uptake in control or ΔN237NLSMondoA expressing HA1E cells. (C) TXNIP and MondoA expression in control and MondoA knockdown HA1ER cells transfected with vector alone or a TXNIP expression vector was determined by Western blotting. (D) Glucose uptake in MondoA knockdown cells with or without overexpression of TXNIP.