Identification of Muscle-specific Biomarkers of Fatty Acid Beta-oxidation

Elevated fat levels within skeletal muscle cells (intramyocellular lipids) are highly correlated with muscle and whole-body insulin resistance, and more prevalent in obesity. The molecular links and metabolic shifts driving this association remain open to debate, but notably, reduced muscle mitochondrial fatty acid (FA) ß-oxidation is more prevalent among insulin-resistant/diabetic persons. Therefore, discovery of biomarkers reflective of the status of an individual's muscle FA ß-oxidation activity or capacity would have tremendous prognostic and diagnostic value in terms of diabetes. Furthermore, characterization of metabolites associated with muscle mitochondrial fat metabolism should uncover candidate signaling factors which tie FA ß-oxidation to insulin signaling. The investigators propose to identify, for the first time, specific biomarkers of muscle FA ß-oxidation using multiple metabolomic analytical platforms to compare metabolite profiles in samples derived from biological systems displaying disparate muscle fat combustion. The current experiment will test whether plasma metabolites and/or metabolite signatures that track efficient muscular FA ß-oxidation can be experimentally increased in obese, insulin-resistant subjects via a diet-exercise regimen designed to improve muscle fitness and FA combustion.

The investigators will use several comprehensive state-of-the-art metabolite analysis platforms to test the hypothesis that specific systemic metabolites or metabolite signatures correlate with myocellular fatty acid combustion. The investigators will then determine whether said metabolites do in fact reflect metabolic health status as predicted, by determining whether profiles are altered in obese subjects whose insulin sensitivity and muscle FA ß-oxidation is improved through aerobic exercise and dietary intervention. Specifically, this study is investigating the usefulness of metabolomic profiles reflective of muscle fat combustion in predicting metabolic health changes following diet \& exercise intervention in obese subjects. The investigators hypothesize that biomarkers reflective of normal, efficient muscle ß-oxidation will be increased, and markers indicative of poor muscle fat combustion reduced, in a cohort of sedentary obese subjects after undergoing a 4 month diet-exercise protocol that will increase muscle fitness and improve insulin action.

In anticipation of 16 subjects completing the study, up to 20 obese female subjects will be recruited from the Davis and Sacramento areas and asked to participate in a weight loss regimen involving caloric restriction (reduction of \~25% of typical caloric intake, with rotating 7-day menus based on nutrient compositions reflective of the U.S. Dietary Guidelines) and regular aerobic exercise designed to target a 10% weight loss over 4 months (Goodpaster et al.,Diabetes 52: 2191-97, 2003). Volunteers to be included in this study will have the following characteristics: age 30-50, BMI between 30-34.9 with stable self-reported body weight over the last 6 mos., sedentary (not participating in a regular exercise program, typical planned exercise ≤30 min. per week assessed by pre-screening telephone questionnaire), non-smoker, no clinical signs of infection (i.e., no fever, CBC wnl), no personal history of cardiovascular disease, no clinically-significant elevations in blood pressure, or diabetes, no regular medications (except oral contraceptives, allergy meds) or drug abuse. These initial studies will involve women only in order to (a) reduce potential sex-related metabolite variability and (b) to better match subject characteristics in previous studies. Each subject will serve as her own control, with changes in metabolite profiles following intervention calculated for each individual. Post-absorptive insulin and glucose measurements will be made to estimate insulin sensitivity by HOMA, and patterns following an OGTT pre- and post-treatment used to assess the insulin sensitivity index using the method of Matsuda et al. (Matsuda \& DeFronzo, Diabetes Care 22:1462-70, 1999). Individual changes in HOMA and insulin sensitivity values comparing pre- and post-intervention periods will be considered to be indicative of changes in insulin action. An acute exercise protocol to elicit muscle work will be carried out over 20 min. to increase metabolic flux of FA in muscle, a strategy designed to enrich the plasma with muscle metabolites. Differences in outcome variables will be tested by repeated-measures ANOVA to determine significance of fitness intervention exercise bout, and intervention exercise bout interactions.

Screening: After signing the Consent Form, an initial fasting blood draw (up to 20 mL) and vital signs will be taken to ensure potential subjects meet the clinical criteria. Height and weight will be determined and interviews conducted to evaluate whether they meet the aforementioned criteria. Body weight will be measured to the nearest 0.1kg with subjects wearing light-weight surgical scrubs. Height will be measured to the nearest 0.1cm using a wall-mounted, Ayrton Stadiometer Model S100 (Ayrton Corporation, Prior Lake, MN). Body Mass Index (BMI) will be calculated as kg/m2. Waist circumference will be measured in the standing position, with measurements obtained midway between the lateral lower rib margin and the iliac crest. The measurement will be taken mid-exhalation, and the average of two readings will be recorded. Hip circumference will be measured at the maximum protuberance of the buttocks. An additional 5 mL of blood will be drawn 2 hours after subjects consume a drink containing a 75 g dose of sugar (an abbreviated oral glucose tolerance test) in order to evaluate insulin sensitivity. A Baecke questionnaire will be used to assess physical activity level to be used in DRI calculations of maintenance calories for the pre-intervention lead-in. To minimize variability in our outcome measures that may be introduced by menstrual cycle hormonal shifts, we will endeavor to maintain consistency by evaluating subjects during the follicular phase of the menstrual cycle (as estimated by cycle records for each woman), preferably beginning on day 4 of the menstrual cycle. Subjects on birth control medication will also preferably be evaluated on day 4 of their menstrual cycle. Subjects will be given a calendar and instructions to record their menstrual cycles throughout the study. Eligible subjects will be asked to visit the WHNRC several times, as follows:

Test Week 1: On day 4 of their menstrual cycle, subjects will come to the clinic for the beginning of Test Week 1. Each day subjects will be given prepared meals to be eaten at the WHNRC, or packed to go along with final preparation instructions. During the test week subjects will receive meals and snacks for a 7 day menu prepared by the WHNRC Metabolic Kitchen and based on U.S. Dietary Guidelines and prescribed to maintain stable body weight. Volunteers will be provided with a daily multivitamin supplement study and will be required to refrain from taking personal dietary supplements or vitamins during the study. This is designed to ensure a similar nutritional profile across all subjects, prior to obtaining biofluids for metabolomic analysis to minimize physiological variability. Subjects will be weighed daily during Test weeks 1 and 2 to ensure body weight maintenance and diet prescriptions adjusted if necessary. During this test week each subject will participate in an instruction session regarding the diet and diet compliance provided under the guidance of a registered dietitian. This instruction may be done individually or in a group setting. Each individual's diet (based on the U.S. Dietary Guidelines) and exercise plan will be designed to elicit a target of 10% body weight loss and to improve fitness over the 16 week intervention period. Test Week 2: Test week 2 duplicates Test Week 1. Subjects will be scheduled based on predictions from their menstrual calendars. This might mean that subjects will continue exercising for additional days in order to coincide with menstrual day 4 when diet is provided and testing begins. The acute exercise will be a 40-50% of the Test week 1 VO2max.

Eligibility Criteria:
Inclusion Criteria:

* female
* age 30-50
* menstruating with regularity
* BMI between 30- 37.5 with stable body weight (\<5% change) over last 6 mos
* waist to hip ratio \>0.9
* sedentary lifestyle (typical exercise \< 30 min. per week)
* non-smoker
* evidence for pre-diabetes (hyperinsulinemia and/or blood glucose \> 100 \< 126 mg/dl)

Exclusion Criteria:

* any clinically significant abnormal laboratory values
* any clinical signs of infection (i.e., fever)
* any chronic diseases
* any personal history of cardiovascular disease
* clinically-significant elevation in blood pressure (≥130/85)
* diabetes or other chronic disease
* any regular medications or drug abuse
* pregnancy or lactation