Mitochondrial ATP synthase as a direct molecular target of chromium(III) to ameliorate hyperglycaemia stress

Abstract

Chromium(III) is extensively used as a supplement for muscle development and the treatment of diabetes mellitus. However, its mode of action, essentiality, and physiological/pharmacological effects have been a subject of scientific debate for over half a century owing to the failure in identifying the molecular targets of Cr(III). Herein, by integrating fluorescence imaging with a proteomic approach, we visualized the Cr(III) proteome being mainly localized in the mitochondria, and subsequently identified and validated eight Cr(III)-binding proteins, which are predominately associated with ATP synthesis. We show that Cr(III) binds to ATP synthase at its beta subunit via the catalytic residues of Thr213/Glu242 and the nucleotide in the active site. Such a binding suppresses ATP synthase activity, leading to the activation of AMPK, improving glucose metabolism, and rescuing mitochondria from hyperglycaemia-induced fragmentation. The mode of action of Cr(III) in cells also holds true in type II diabetic male mice. Through this study, we resolve the long-standing question of how Cr(III) ameliorates hyperglycaemia stress at the molecular level, opening a new horizon for further exploration of the pharmacological effects of Cr(III).

Fig. 1. Mining Cr³⁺-associated targets through fluorescent labelling.

a Structure of Cr³⁺-NTA-AC. b Representative confocal microscopic imaging of live HepG2 cells under high glucose stress (40 mM) labelled with 50 μM Cr³⁺-NTA-AC and MitoTracker red (n = 5), demonstrating that most of the blue signals localized in mitochondria. Scale bar: 10 μm. c Selected Cr³⁺-associated proteins in HepG2 cells identified in 2-DE by fluorescence imaging and silver staining. Full 2-DE images are shown in Supplementary Fig. 2. d The protein-protein interaction (PPI) network of identified Cr³⁺-associated proteins in HepG2 cells generated via STRING. The PPI network of Cr³⁺-associated proteins was exported from the STRING database (www.string-db.org). e Representative SDS-PAGE of ATP5B with Coomassie Blue staining and fluorescence image. n = 3; mean ± SEM. Two-sided Student’s t test. f Representative SDS-PAGE of ATP5L with Coomassie Blue staining and fluorescence image. n = 3; mean ± SEM. Two-sided Student’s t test. g Cellular thermal-shift assays (CETSA) demonstrating the binding of Cr³⁺ to, ATP5L, ATP5B and Hsp60 in cellulo. n = 3; mean ± SEM. One representative result of three independent experiments is shown (b, c). Source data are provided as a Source Data file.

Fig. 2. Cr³⁺ attenuates ATP synthase activity and activates AMPK through binding to ATP5B catalytic site.

a Dose-dependent inhibition of ATP synthase activity by Cr³⁺ treatment in HepG2 cells under hyperglycaemia condition. ATP5B inhibitor Octyl-α-ketoglutarate (O-KG) is used as a control. n = 3; mean ± SEM. Two-sided Student’s t test. b Time-dependent inhibition of ATP synthase activity by Cr³⁺ treatment in HepG2 cells under hyperglycaemia condition. n = 3; mean ± SEM. Two-sided Student’s t test. c AMP/ATP ratios in HepG2 cells under different conditions. n = 6; mean ± SEM. Two-sided Student’s t test. d Dose-dependent and (e) Time-dependent activation of AMPK and ACC by Cr³⁺ in HepG2 cells under hyperglycaemia condition. n = 3; mean ± SEM. f The effect of ATP5B gene silencing on Cr³⁺-mediated AMPK and ACC activation in HepG2 cells. n = 3; mean ± SEM. Two-sided Student’s t test. g The substitution of Mg²⁺ in ATP5B by Cr³⁺ as determined by ICP-MS. n = 3; mean ± SEM. h The metal contents in Cr-ATP5B upon supplementation of various amounts of Mg²⁺. n = 3; mean ± SEM. i The Cr³⁺ binding capability of wild-type (WT) ATP5B and various mutants as determined by ICP-MS. n = 3; mean ± SEM. Two-sided Student’s t test. GAPDH is used as a control. Source data are provided as a Source Data file.

Fig. 3. Cr³⁺ rescues mitochondria from hyperglycaemia-induced fragmentation through targeting ATP5B.

a Representative microscopic imaging of HepG2 cells with mitochondria stained by MitoTracker under normal glucose condition (5.6 mM, as Ctrl), high glucose-induced stress (40 mM, as HG) and high glucose-induced stress with Cr³⁺ supplementation (100 µM) (HG + Cr³⁺) (n = 10). Inset: 4 times enlargement of mitochondria. Scale bars: 5 µm. b, c Quantitative analysis and quantification of mitochondrial morphology illustrated by form factor (FF) and aspect ratio (AR). n = 10; mean ± SEM. Two-sided Student’s t test. d, e Quantitative analysis and quantification of mitochondrial morphology in scramble or ATP5B null (siATP5B-transfected) HepG2 cells. n = 5; mean ± SEM. Two-sided Student’s t test. Representative microscopic images are shown in Supplementary Fig. 11. Mitochondria remains fragmented even when 100 µM Cr³⁺ were supplemented to ATP5B-silenced cells. Individual data points (c, e) are available from Source Data, which are provided as a Source Data file.

Fig. 4. Cr³⁺ ameliorates hyperglycaemia stress through targeting ATP synthase in cells and diabetic mice.

a Comparison of glucose consumption in HepG2 cells. n = 3; mean ± SEM. Two-sided Student’s t test. Representative western blot (b) and quantification results (c) showing the effect of ATP5B gene silencing on Cr³⁺-mediated regulation of glycolysis and glycogenesis enzymes in HepG2 cells. n = 3; mean ± SEM. Two-sided Student’s t test. d Schematic illustration of animal study. e Blood glucose levels in db/db and wild-type (WT) mice with or without treatment of Cr³⁺ (n = 6). f Cr³⁺ contents in the livers of both db/db and wild-type (WT) mice with or without treatment of Cr³⁺ (n = 6). g Cr³⁺ inhibits ATP synthase activity in db/db mice (n = 6). h AMP/ATP ratios in the livers of db/db and WT mice with or without Cr³⁺ treatment (n = 6). For (f–h), centre line, median; box limits, upper and lower boundary, 75 and 25% interquartile ranges respectively; whiskers, maxima and minima; points, all data points. Two-sided Student’s t test. i Cr³⁺ activates AMPK and ACC in db/db mice. n = 6; mean ± SEM. Two-sided Student’s t test. j Proposed scheme showing that Cr³⁺ ameliorates hyperglycaemia stress through inhibition of ATP synthase and subsequent activation of AMPK in diabetic mice. Source data are provided as a Source Data file.