MOLECULAR REGULATION OF MUSCLE GLUCOSE METABOLISM (AIMS 2A, 2B, 3)
MOLECULAR REGULATION OF MUSCLE GLUCOSE METABOLISM (AIMS 2A, 2B, 3)
The molecular nature of insulin resistance in human muscle is still incompletely defined. Our
 data indicate that acetylation of mitochondrial proteins in humans is regulated by muscle
 contraction and is dysregulated in insulin resistance. Poor function of mitochondria in
 skeletal muscle is a hallmark of insulin resistance in skeletal muscle. We propose to use a
 combination of clinical research and mass spectrometry techniques to determine how the
 cytosolic and mitochondrial protein acetylation is regulated by muscle contraction in insulin
 sensitive and resistant human volunteers. We will test the hypothesis that mitochondrial
 protein acetylation is decreased to a greater degree following a bout of exercise in insulin
 sensitive than in insulin resistant human muscle. Using these techniques we also propose to
 determine how acetylation of mitochondrial adenine nucleotide translocase (ANT1) at lysines
 10, 23, and 92 regulates ANT1 structure and function. Finally, we propose 4) to use a
 combination of molecular modeling and in vitro assays together with the approach developed in
 Aim 3 to characterize the role of acetylation in other mitochondrial proteins. Protein
 targets for this aim will be prioritized based on the potential role of the protein in
 insulin resistance or mitochondrial function as well as dysregulation of its acetylation
 state in insulin resistant muscle.
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.
Inclusion Criteria:

 -  Aim 2a. Subjects. Two groups (aged 21-65) will be studied: lean (BMI<25), healthy
 insulin sensitive subjects, and obese (BMI>30) nondiabetics 20 subjects will be
 enrolled

 1. Subjects must be able to communicate meaningfully with the investigator and must
 be legally competent to provide written informed consent.

 2. Subjects may be of either sex with age as described in each protocol. Female
 subjects must be non-lactating and will be eligible only if they have a negative
 pregnancy test throughout the study period.

 3. Subjects must range in age as described in each specific protocol.

 4. Subjects must have the following laboratory values:

 Hematocrit ≥ 35 vol% Serum creatinine ≤ 1.6 mg/dl AST (SGOT) < 2 times upper
 limit of normal ALT (SGPT) < 2 times upper limit of normal Alkaline phosphatase <
 2 times upper limit of normal Triglycerides < 150 mg/dl. PT 11.7 -14.3 (During
 Liposyn/heparin infusion, PT will be determined to insure that it is < 1.5-2.0
 times the normal value.) PTT 23.0-37.0.

 Aim 2b (muscle contraction and acetylation). Subjects. Two groups (aged 21-65)
 will be studied: lean (BMI<25), healthy insulin sensitive subjects, and obese
 (BMI>30) 32 subjects will be enrolled

 Inclusion Criteria

 -  Subjects must be able to communicate meaningfully with the investigator and
 must be legally competent to provide written informed consent.

 -  Subjects may be of either sex with age as described in each protocol. Female
 subjects must be non-lactating and will be eligible only if they have a
 negative pregnancy test throughout the study period.

 -  Subjects must range in age as described in each specific protocol.

 -  Subjects must have the following laboratory values:

 Hematocrit ≥ 35 vol% Serum creatinine ≤ 1.6 mg/dl AST (SGOT) < 2 times upper
 limit of normal ALT (SGPT) < 2 times upper limit of normal Alkaline phosphatase <
 2 times upper limit of normal Triglycerides < 150 mg/dl. PT 11.7 -14.3 (During
 Liposyn/heparin infusion, PT will be determined to insure that it is < 1.5-2.0
 times the normal value.) PTT 23.0-37.0.

 Aim 3 Subjects. Two groups (aged 21-65) will be studied: lean (BMI<25), healthy
 insulin sensitive subjects, and obese (BMI>30) 20 subjects will be enrolled.

 Inclusion Criteria

 -  Subjects must be able to communicate meaningfully with the investigator and
 must be legally competent to provide written informed consent.

 -  Subjects may be of either sex with age as described in each protocol. Female
 subjects must be non-lactating and will be eligible only if they have a
 negative pregnancy test throughout the study period.

 -  Subjects must range in age as described in each specific protocol.

 -  Subjects must have the following laboratory values:

 Hematocrit ≥ 35 vol% Serum creatinine ≤ 1.6 mg/dl AST (SGOT) < 2 times upper
 limit of normal ALT (SGPT) < 2 times upper limit of normal Alkaline phosphatase <
 2 times upper limit of normal Triglycerides < 150 mg/dl. INR < 1.3

 Exclusion Criteria:

 -  Aim 2a

 -  Subjects must not be receiving any of the following medications: thiazide or
 furosemide diuretics, beta-blockers, or other chronic medications with known
 adverse effects on glucose tolerance levels unless the patient has been on a
 stable dose of such agents for the past three months before entry into the study.
 Subjects may be taking a stable dose of estrogens or other hormonal replacement
 therapy, if the subject has been on these agents for the prior three months.
 Subjects taking systemic glucocorticoids are excluded.

 -  Subjects with a history of clinically significant heart disease (New York Heart
 Classification greater than grade II; more than non-specific ST-T wave changes on
 the EKG), peripheral vascular disease (history of claudication), or pulmonary
 disease (dyspnea on exertion of one flight or less; abnormal breath sounds on
 auscultation) will not be studied.

 -  Recent systemic or pulmonary embolus, untreated high-risk proliferative
 retinopathy, recent retinal hemorrhage, uncontrolled hypertension, systolic
 BP>180, diastolic BP>105, autonomic neuropathy, resting heart rate >100,
 electrolyte abnormalities.

 Aim 2b Exclusion Criteria

 -  Subjects must not be receiving any of the following medications: thiazide or
 furosemide diuretics, beta-blockers, or other chronic medications with known adverse
 effects on glucose tolerance levels unless the patient has been on a stable dose of
 such agents for the past three months before entry into the study. Subjects may be
 taking a stable dose of estrogens or other hormonal replacement therapy, if the
 subject has been on these agents for the prior three months. Subjects taking systemic
 glucocorticoids are excluded.

 -  Subjects with a history of clinically significant heart disease (New York Heart
 Classification greater than grade II; more than non-specific ST-T wave changes on the
 EKG), peripheral vascular disease (history of claudication), or pulmonary disease
 (dyspnea on exertion of one flight or less; abnormal breath sounds on auscultation)
 will not be studied.

 -  Recent systemic or pulmonary embolus, untreated high-risk proliferative retinopathy,
 recent retinal hemorrhage, uncontrolled hypertension, systolic BP>180, diastolic
 BP>105, autonomic neuropathy, resting heart rate >100, electrolyte abnormalities.

 Aim 3 Exclusion Criteria

 -  Subjects must not be receiving any of the following medications: thiazide or
 furosemide diuretics, beta-blockers, or other chronic medications with known adverse
 effects on glucose tolerance levels unless the patient has been on a stable dose of
 such agents for the past three months before entry into the study. Subjects may be
 taking a stable dose of estrogens or other hormonal replacement therapy, if the
 subject has been on these agents for the prior three months. Subjects taking systemic
 glucocorticoids are excluded.

 -  Subjects with a history of clinically significant heart disease (New York Heart
 Classification greater than grade II; more than non-specific ST-T wave changes on the
 EKG), peripheral vascular disease (history of claudication), or pulmonary disease
 (dyspnea on exertion of one flight or less; abnormal breath sounds on auscultation)
 will not be studied.

 -  Recent systemic or pulmonary embolus, untreated high-risk proliferative retinopathy,
 recent retinal hemorrhage, uncontrolled hypertension, systolic BP>180, diastolic
 BP>105, autonomic neuropathy, resting heart rate >100, electrolyte abnormalities.

 -  Healthy controls with BMI less than 25 who have first degree relatives with Type 2
 diabetes are at high risk for insulin resistance, this is an exclusion for this study.