AMPK activation counters hepatic glucose production, muscle insulin resistance and hyperlipidemia in diabetes. Metformin may indirectly target AMPK, but its mechanism is debated. No other diabetes drug effectively targets AMPK. Given that many xenobiotics reportedly influence AMPK, and salicylate was recently found to activate it after centuries of use as anti-inflammatory drug, we reasoned that other drugs in current practice may also activate AMPK. Xanthine oxidase (XO) inhibitors are used to treat gout by lowering uric acid levels. Following fundamental biochemical principles, we reasoned that blocking the final steps in purine metabolism would increase concentrations of purine metabolites upstream of the blockade, including AMP and AICAR, which would activate AMPK. Preliminary results show this is true. LC-MS/MS based metabolomics studies, conducted with livers from XOI-treated HFD mice and cultured hepatocytes, show that both allopurinol and febuxostat decrease intracellular uric acid and xanthine and concomitantly increase hypoxanthine, IMP, AMP and AICAR. The metabolomics profiles also revealed changes readily linked to AMPK activation. AMPK activation was also demonstrated by appropriate phosphorylations within AMPK and its substrates, ACC and Raptor. Consistent with AMPK activation, the XOIs lowered blood glucose and insulin, reduced hepatic TG, and improved glucose and insulin tolerance in obese mice, without affecting body weight or food intake. And since purine repletion restored metabolic homeostasis to the obese mice, we reasoned that obesity might be a state of purine depletion leading to dysregulated AMPK. This too is supported by preliminary results and many previous reports in the literature, even though the physiology and pathology of XO and AMPK have not been linked previously. Proposed experiments expand the studies to adipose tissue, muscle, intestine, and the vasculature using genetic modifications or viral transduction to acutely up/down regulate XO and AMPK in selected tissues of obese mice. The modified mice and derived cells will be treated with XOIs. Metabolomics studies will document concentrations of purine and AMPK-relevant metabolites, and XO and AMPK activities will be demonstrated biochemically and through protein phosphorylation and gene expression. The in vivo metabolic and cardiovascular effects of these manipulations will be assessed using standard methods, including but not limited to glucose and insulin tolerance tests and blood concentrations, hyperinsulinemic-euglycemic clamps, tissue triglyceride content and histopathology, and quantification and composition of vascular plaque. This proposal addresses two major questions/hypotheses: 1) Does the increased XO activity in tissues of obese animals (possibly occurring secondary to inflammation) promote purine depletion to suppress AMPK and adversely impact cardiometabolic homeostasis? 2) Does XO inhibition replete tissue purines to promote AMPK activation and restore cardiometabolic homeostasis? Proposed preclinical experiments test these hypotheses in preparation for clinical translation.

Public Health Relevance

We have found that drugs used to treat gout also raise tissue purines to activate AMPK, a favored target in diabetes, and this is associated with glucose lowering and other metabolic improvements in diabetic mice. Proposed studies expand on this by asking whether reduced tissue purines in obesity inactivate AMPK to dysregulate metabolic homeostasis and promote diabetes and cardiovascular risk.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK108936-01
Application #
9083155
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Pawlyk, Aaron C
Project Start
2016-05-16
Project End
2019-03-31
Budget Start
2016-05-16
Budget End
2017-03-31
Support Year
1
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Joslin Diabetes Center
Department
Type
DUNS #
071723084
City
Boston
State
MA
Country
United States
Zip Code
Goldfine, Allison B; Shoelson, Steven E (2017) Therapeutic approaches targeting inflammation for diabetes and associated cardiovascular risk. J Clin Invest 127:83-93