Project Grant R21AG084931

NON-CANONICAL MECHANISMS AND UNCONVENTIONAL APPROACHES TO ACTIVATE AMPK AND GLUCOSE UPTAKE IN THE SKELETAL MUSCLE OF OLDER RATS - AMP-ACTIVATED PROTEIN KINASE (AMPK) IS A HIGHLY CONSERVED SERINE/THREONINE KINASE WITH MANY IMPORTANT EFFECTS, INCLUDING PUTATIVE ANTI-AGING ACTIONS. AMPK PLAYS A KEY ROLE IN THE CONTROL OF GLUCOSE UPTAKE BY SKELETAL MUSCLE, THE TISSUE THAT ACCOUNTS FOR UP TO 85% OF INSULIN-INDUCED BLOOD GLUCOSE CLEARANCE. INSULIN RESISTANCE FOR MUSCLE GLUCOSE UPTAKE IS AN ESSENTIAL DEFECT FOR TYPE 2 DIABETES AND LINKED TO MANY OF THE MOST PREVALENT AND DEVASTATING AGE-RELATED PATHOLOGIES, INCLUDING CARDIOVASCULAR DISEASE AND COGNITIVE DYSFUNCTION. CALORIC RESTRICTION (CR; REDUCING CALORIE INTAKE 20-40% BELOW AD LIBITUM INTAKE) ENHANCES INSULIN-STIMULATED GLUCOSE UPTAKE IN MUSCLE OF OLD RATS. THE BROAD, LONG-TERM GOAL OF THIS PROJECT IS TO ADVANCE UNDERSTANDING OF MECHANISMS TO IMPROVE INSULIN SENSITIVITY, LEADING TO HEALTHY AGING. THE SPECIFIC AIMS ARE: 1) TO DETERMINE CR EFFECTS ON KEY ALLOSTERIC REGULATORS OF AMPK, AND ON THE ACTIVATION OF AMPK IN SELECTED SUBCELLULAR COMPARTMENTS OF SKELETAL MUSCLE FROM OLDER RATS; AND 2) TO ASCERTAIN ALDOMETANIB (A RECENTLY DISCOVERED AMPK ACTIVATOR) EFFECTS ON OLDER RAT SKELETAL MUSCLE SUBCELLULAR LOCALIZATION OF AMPK T172 PHOSPHORYLATION, GLUCOSE UPTAKE, CELL SURFACE GLUT4 LOCALIZATION, AND KEY SIGNALING PROTEINS. CR BY OLDER RATS LEADS TO GREATER PHOSPHORYLATION OF AMPK ON THE KEY REGULATORY T172 SITE (PAMPKTHR172) CONCOMITANT WITH GREATER PHOSPHORYLATION OF MULTIPLE AMPK SUBSTRATES IN MUSCLE INDEPENDENT OF CHANGES IN MUSCLE ATP OR AMP, THE MAJOR ALLOSTERIC REGULATORS OF AMPK. BECAUSE CONVENTIONAL MECHANISMS CANNOT EXPLAIN THESE STRIKING RESULTS, THIS PROJECT WILL PROBE NON-CANONICAL MECHANISMS, INCLUDING THE MUSCLE CONCENTRATION OF FRUCTOSE-1, 6- BISPHOSPHATE THAT INCREASES PAMPKTHR172 BY AN AMP-INDEPENDENT MECHANISM. AMPK RESIDES IN MULTIPLE COMPARTMENTALIZED POOLS, AND SPATIAL REGULATION OF AMPK IS A CRUCIAL DETERMINANT OF FUNCTION, YET NO EARLIER CR RESEARCH ANALYZED COMPARTMENTALIZED AMPK ACTIVATION. ACCORDINGLY, CR EFFECTS ON SUBCELLULAR LOCALIZATION OF PAMPKTHR172 AND PHOSPHORYLATION OF AMPK SUBSTRATES WILL BE EVALUATED. ALTHOUGH CR IMPROVES INSULIN SENSITIVITY, ITS WIDESPREAD ADOPTION BY HUMANS IS UNREALISTIC. IDENTIFICATION OF SAFE AND EFFECTIVE ALTERNATIVE INTERVENTIONS IS ESSENTIAL. A NOVEL ALDOLASE INHIBITOR (ALDOMETANIB) SELECTIVELY INCREASED LYSOSOMAL PAMPKTHR172 WITHOUT ALTERING PAMPKTHR172 IN OTHER SUBCELLULAR LOCATIONS, AND ALSO WITHOUT ALTERING THE CONCENTRATIONS OF AMP OR ATP IN CULTURED CELLS. BECAUSE THE SPECIFIC MECHANISMS WHEREBY ALDOMETANIB ELEVATES GLUCOSE UPTAKE ARE UNKNOWN, THIS PROJECT WILL DETERMINE ALDOMETANIB'S EFFECTS ON SUBCELLULAR LOCALIZATION OF PAMPKTHR172, GLUT4 TRANSPORTER TRANSLOCATION, AND REGULATION OF KEY SIGNALING PROTEINS THAT REGULATE GLUCOSE UPTAKE IN THE SKELETAL MUSCLE OF OLDER RATS. THIS STUDY WILL CHALLENGE CONVENTIONAL WISDOM ABOUT CR'S REGULATION OF AMPK, AND REVEAL VALUABLE INSIGHTS INTO A PROMISING, NEW AMPK ACTIVATOR'S MECHANISMS AND ACTIONS ON MUSCLE GLUCOSE UPTAKE.