MOLECULAR REGULATION OF MUSCLE GLUCOSE METABOLISM (AIMS 2A, 2B, 3)

The purpose of this project has been to define the molecular basis for insulin resistance in human muscle. The preceding two cycles of this project were devoted to using proteomics approaches to map and quantify serine/threonine (S/T) phosphorylation sites on insulin receptor substrate (IRS)-1 in skeletal muscle from healthy and insulin resistant humans. Recent proteomics experiments reveal that another protein modification, lysine acetylation, is more common in non-histone proteins than has been recognized. There are no data, to our knowledge, regarding a relationship between insulin resistance in vivo in human muscle and protein acetylation. We have used proteomics techniques to discover a number of lysine acetylation sites in skeletal muscle proteins in vivo in humans, and this is prominent in mitochondrial proteins. Preliminary data show that mitochondrial protein acetylation is regulated by muscle contraction, and that this response is reduced in insulin resistance. Finally, the mitochondrial inner membrane protein adenine nucleotide translocase (ANT)1 was abundantly acetylated under resting conditions, and its deacetylation in response to contraction was reduced in insulin resistance. Because ANT1 exerts significant physiological control strength for ADP/ATP exchange, and because mitochondrial function is altered in insulin resistance, it is important to characterize this abnormality. Therefore, in order to determine how abnormalities in regulation of lysine acetylation characterize insulin resistant muscle, we propose: 2\. To determine how the cytosolic and mitochondrial protein acetylomes are regulated by muscle contraction in insulin sensitive and resistant human volunteers. We will test the hypotheses that: a. There is differential acetylation of cytosolic or mitochondrial proteins in insulin resistance. b. Mitochondrial protein acetylation is decreased to a greater degree following a bout of exercise in insulin sensitive than in insulin resistant human muscle. 3\. To determine how acetylation of the mitochondrial inner membrane adenine nucleotide translocase ANT1 regulates protein structure and function. We will use mitochondria isolated from human muscle biopsies and molecular dynamics and elastic network modeling to test the hypotheses that: 1. Deacetylation of lysines 10, 23, and 92 increases ANT1 activity. 2. Oxidation of the mitochondrial matrix (increasing the NAD/NADH ratio) raises deacetylation activity and decreases acetylation of ANT1. 3. ANT1 molecular dynamics are altered by acetylation of lysines 10, 23, and 92. These aims that involve clinical research will be complemented by two aims (Aims 1 and 4) performed entirely in vitro, not using human specimens or data. For context, these aims are listed on page 1 of the accompanying Research Proposal. We have retained the numbering scheme from the original proposal to avoid confusion. Please note that only Aims 2 and 3 involve clinical research.