AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function
Laura L. Dugan, … , Robert K. Naviaux, Kumar Sharma
Laura L. Dugan, … , Robert K. Naviaux, Kumar Sharma
Published November 1, 2013; First published October 25, 2013
Citation Information: J Clin Invest. 2013;123(11):4888-4899. https://doi.org/10.1172/JCI66218.
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Research Article

AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function

Abstract

Diabetic microvascular complications have been considered to be mediated by a glucose-driven increase in mitochondrial superoxide anion production. Here, we report that superoxide production was reduced in the kidneys of a steptozotocin-induced mouse model of type 1 diabetes, as assessed by in vivo real-time transcutaneous fluorescence, confocal microscopy, and electron paramagnetic resonance analysis. Reduction of mitochondrial biogenesis and phosphorylation of pyruvate dehydrogenase (PDH) were observed in kidneys from diabetic mice. These observations were consistent with an overall reduction of mitochondrial glucose oxidation. Activity of AMPK, the major energy-sensing enzyme, was reduced in kidneys from both diabetic mice and humans. Mitochondrial biogenesis, PDH activity, and mitochondrial complex activity were rescued by treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). AICAR treatment induced superoxide production and was linked with glomerular matrix and albuminuria reduction in the diabetic kidney. Furthermore, diabetic heterozygous superoxide dismutase 2 (Sod2+/–) mice had no evidence of increased renal disease, and Ampka2–/– mice had increased albuminuria that was not reduced with AICAR treatment. Reduction of mitochondrial superoxide production with rotenone was sufficient to reduce AMPK phosphorylation in mouse kidneys. Taken together, these results demonstrate that diabetic kidneys have reduced superoxide and mitochondrial biogenesis and activation of AMPK enhances superoxide production and mitochondrial function while reducing disease activity.

Authors

Laura L. Dugan, Young-Hyun You, Sameh S. Ali, Maggie Diamond-Stanic, Satoshi Miyamoto, Anne-Emilie DeCleves, Aleksander Andreyev, Tammy Quach, San Ly, Grigory Shekhtman, William Nguyen, Andre Chepetan, Thuy P. Le, Lin Wang, Ming Xu, Kacie P. Paik, Agnes Fogo, Benoit Viollet, Anne Murphy, Frank Brosius, Robert K. Naviaux, Kumar Sharma

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Figure 2

EPR spectra and ROS release rate in control and diabetic kidney mitochondria.

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EPR spectra and ROS release rate in control and diabetic kidney mitochon...
Dissociated kidney cell suspensions from control (A) or diabetic (B) animals were prepared as described and analyzed by EPR using DPMPO as the spin probe 10 minutes after addition of the mitochondrial substrates malate and pyruvate. The characteristic spectrum for the superoxide-DPMPO was observed in both control and diabetic samples at baseline. However, addition of high glucose (bottom traces) did not increase mitochondrial superoxide production and, in the diabetic kidney samples, actually reduced superoxide production. (C) Analysis of the EPR signal intensity using the upfield peak at 334 mT, normalized to mg protein. Values are mean ± SEM, n = 6 per condition, *P < 0.05 vs control. The second set of bars to the right show data derived from Supplemental Figure 3, which shows additional spectra from samples after addition of the mitochondrial complex III inhibitor, antimycin A. Antimycin A produced the expected increase in superoxide, confirming the ability of this technique to detect increased mitochondrial superoxide production if present. (D) Decreased ROS release from kidney mitochondria isolated from diabetic animals Measurements were made in the presence of succinate (Succ), succinate and FCCP (Succ + FCCP), glutamate plus malate (G/M) or glutamate plus malate with rotenone (G/M + Rot) by Amplex Red assay. Rates were calculated using calibration curve as described in Methods. Values are mean ± SEM, n = 5–8. *P < 0.001 vs. corresponding control; #P < 0.05 vs. G/M.