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Department of Nutritional Sciences (I.E.L., K.L.H., K.L.W., T.L.Z., S.I.K., M.L.F.), University of Connecticut, Storrs, Connecticut
School of Allied Health (R.A.B., J.Z.I.), University of Connecticut, Storrs, Connecticut
Department of Animal & Nutritional Sciences, The University of New Hampshire, Durham, New Hampshire (I.E.L.)
Address reprint requests to: Maria Luz Fernandez, PhD, University of Connecticut, Department of Nutritional Sciences 3624 Horsebarn Road Extension, U 4017, Storrs, CT 06269. E-mail: maria-luz.fernandez{at}uconn.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSREFERENCES |
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Methods: Participants consumed a hypocaloric diet; 30% protein, 30% fat and 40% carbohydrate in addition to increasing number of steps/day. Carnitine supplementation followed a randomized double blind protocol. Protocol lasted for 10 weeks. Seventy subjects (35 in the control and 35 in the carnitine group) completed the intervention. Anthropometrics, plasma insulin and leptin concentrations and body composition were measured. The number of subjects with the MetSyn and insulin resistance, were assessed at baseline and post-intervention.
Results: Because there were no significant differences between the carnitine and the placebo groups for all measured parameters, participants were grouped together for all analysis. Subjects decreased total energy (–26.6%, p < 0.01) and energy from carbohydrate (–17.3%, p < 0.01) and increased energy from protein by 67% (p < 0.01) and number of steps/day (42.6%, p < 0.01). Body weight (–4.6%, p < 0.001), body mass index (–4.5%, p < 0.01), waist circumference (–6.5%, p < 0.01), total fat mass (–1.7%, p < 0.01), trunk fat mass (–2.0%, p < 0.01), insulin (– 17.9%, p < 0.01) and leptin (–5.9%, p < 0.05) decreased after the intervention. Ten of 19 participants with insulin resistance became insulin sensitive and 7 of 8 participants with the MetSyn no longer had the syndrome after the intervention.
Conclusion: Moderate increases in physical activity and a hypocaloric/high protein diet resulted in multiple beneficial effects on body anthropometrics and insulin sensitivity. Realistic dietary and physical activity goals must be the focus of intervention strategies for overweight and obese individuals.
Key words: premenopausal women, body composition, weight loss, insulin resistance, metabolic syndrome
Abbreviations: CHD=coronary heart disease • CPT-1=carnitine palmitoyltransferase • DM2=diabetes mellitus type 2 • DXA=dual X-ray absorption • FFQ=food frequency questionnaire • HDL-C=HDL cholesterol • IPAQ=international physical activity questionnaire • LCFA=long chain fatty acid • LDL-C=LDL-cholesterol • MS=metabolic syndrome • NDS=nutrient database system • TG-triglycerides
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSREFERENCES |
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Both insulin, a regulator of lipogenesis/lipolysis [10], and leptin, a regulator of energy expenditure and appetite control [8], are typically elevated in obesity [11] and decrease with weight loss [12]. Evidence has shown that decreased percent body fat and insulin levels may increase lipolysis, resulting in increased fatty acid utilization for energy [10]. In addition, active muscle fibers utilize circulating free fatty acids and muscle TG for energy [13]. Therefore, combining calorie restriction with increased physical activity potentially enhances fat oxidation and decreases fat mass. Other mechanisms that could enhance fat oxidation are also intriguing. For example, carnitine is an integral component in long chain fatty acid (LCFA) transport in the mitochondria for ß-oxidation [14]. Supplemental carnitine has been shown to increase weight loss in animals [15] and increase LCFA oxidation in adults (3g carnitine/day) [16]. However, previous studies utilizing 2 to 5 g/day of supplemental carnitine yield inconsistent results [17]. For example, Villani et al did not find changes in total body mass or fat mass in moderately obese women after providing 2 g carnitine/day but did report an increase in resting energy expenditure [18].
Because of the myriad of problems associated with obesity, including insulin resistance, diabetes mellitus type 2 (DM2), the metabolic syndrome (MS), and CHD, weight loss interventions must target multiple risk factors. These risk factors include body composition, lipid profile, and hormonal milicu. The present study was performed to explore the impact of a weight loss program, including dietary modification, increased physical activity, and carnitine supplementation, on anthropometric and body composition measures and plasma insulin and leptin concentrations. We hypothesized that weight loss would favorably affect those parameters associated with the MS and insulin resistance. We also hypothesized that carnitine supplementation would enhance the intervention effects.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSREFERENCES |
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Study Population
Eighty-five overweight/obese, premenopausal women were recruited from the University and surrounding communities. The seventy women (74% Caucasian) who completed the protocol were between 20 and 45 years of age and had a BMI between 25 to 37 kg/m2. Subjects were excluded if they were pregnant, lactating, or had a history of CHD, diabetes, kidney or liver disease. Using the International Physical Activity Questionnaire (IPAQ) [19], the majority of participants considered themselves to be sedentary to moderately active.
Experimental Design
The 10-week study protocol was approved by the University of Connecticut Institutional Review Board. Informed consent was given by all participants. The first component, dietary modification, entailed caloric restriction and modified macronutrient composition. Caloric need was estimated from the Harris-Benedict formula (utilized because of ease of use in the clinical setting), and multiplied by a low activity factor of 1.2. The expected energy distribution of the diet during the protocol was 30% protein, 30% fat, and 40% carbohydrate. Carbohydrate consumption was decreased to prevent inhibition from the first carnitine-dependent LCFA transport protein, carnitine palmitoyltransferase I (CPT-I) [14, 20]. However, a moderate carbohydrate restriction (40%) was chosen to help maintain dietary compliance. Participants received menus with expected caloric level and macronutrient composition. In addition, participants received groceries specified in the menus except for skim milk, condiments, butter or margarine. The second intervention component was increased physical activity as monitored by a pedometer. The third component, carnitine supplementation, utilized a double-blind, randomization to active (3 grams/day of carnitine) or placebo (3 grams/day of cellulose) pills.
On day zero, subjects were assigned a calorie level, and received a pedometer and the menus, groceries, and supplement for that week. They were instructed to record any deviations from the menu and to consume three active or placebo pills with breakfast and lunch. Log sheets were provided to record daily supplement intake. Subjects continued collecting groceries, menus, and supplements weekly.
Two separate fasting (12 h) blood samples were collected during weeks 1, 5, and 10. Blood was collected into tubes containing 0.15 g/100 g EDTA. Plasma was separated by centrifugation at 1,500 x g for 20 min at 4°C, and placed into vials containing PMSF (0.05 g/100g), sodium azide (0.01g/100g) and aprotonin (0.01g/100g). Samples were then placed into –80° freezer until analysis. Plasma samples were used to determine plasma lipids and hormone concentrations.
Dietary Assessment
Baseline dietary data was collected utilizing a 120 item food frequency questionnaire (FFQ) developed by the Fred Hutchinson Cancer Research Center (Seattle, Washington). One participant did not have a complete FFQ and it was not included in the analysis. Participants recorded how many times, on average, in the past three months they had consumed each food listed. Participants selected serving size based on pictures of small, medium, and large food items provided with the FFQ. Menus from 3 non-consecutive weeks during the intervention period (15 weekdays and 6 weekend days) were entered into the Nutrient Database Systems (NDS) for Research, version 4.05_33 (Nutrition Coordinating Center, University of Minnesota) for analysis. Using NDS data, compliance with caloric restriction and macronutrient composition was evaluated.
Physical Activity Assessment
Participants completed the IPAQ (short version, 4 items) on day zero to determine baseline activity. This IPAQ has been shown to collect reliable and valid data on physical activity [19]. During the first week, baseline number of steps was determined utilizing Omron HJ-104 pedometers (Omron Healthcare, Inc., Vernon Hills, IL). Each pedometer was calibrated to individual participant’s stride according to the manufacturer’s instructions. Briefly, stride length was determined from the amount of ground covered in 10 strides at the same pre-measured spot. Pedometers were calibrated at least one other time during the protocol. The pedometer counts the number of steps/24 h period, after which, it resets to zero. Standard deviation is ± 5% (personal communication with Omron Healthcare). This pedometer also has a 7-day memory function. Daily log sheets were collected from participants weekly. Subjects were selected at random to verify the reported number of steps/day compared to values saved in the 7 day memory. At the beginning of week 2, participants were instructed to walk 1500 additional steps than those at baseline. The steps/day goal was increased by 1500 steps two additional times at weeks 4 and 6 for a total increase of 4500 steps/day.
Anthropomentric Measurements
Weight was measured to the closest 0.5 lb and height was measured to the closest 0.5 inch on a portable stadiometer/scale [21]. Weight and height were converted into metric measures to calculate BMI (kg/m2). Waist circumference (WC) was measured midway between the lowest rib and iliac crest to the nearest 0.1 cm [21, 22]. Hip circumference was measured at the widest point on the hip. These measurements were utilized for the waist to hip ratio (WHR). Blood pressure was measured on the right arm using a Welch Allyn, Tycos blood pressure cuff (Welch Allyn, Arden, North Carolina) with the participant seated, following a 5-minute rest.
Body Composition
Body composition was obtained utilizing dual X-ray absorption (DXA) with a Lunar DPX-MD machine (GE Medical Systems, Madison, WI). DXA is a reliable technology for body composition in research [23] and data suggest that it has better reproducibility than skinfold thickness, bioelectrical impedance [24], and near infrared interactance [25] in obese women. DXA scans and analysis were performed in the Bone and Mineral Metabolism Lab at the University of Connecticut. The upper weight limit for the table was 114 kg and subjects were positioned to fit completely within the scanning area. Quality assurance of the DXA machine was performed daily. The coefficients of variation for this study were 0.629% and 0.268% for percent of total and trunk fat and 0.567% and 0.916% for grams of total and trunk fat.
Plasma Glucose, Insulin and Leptin
Plasma glucose was determined enzymatically using Wako kits (Wako Chemicals USA, Richmond, VA) [26]. Plasma insulin was measured using Linco RIA kits (Linco Research, Inc, St. Charles, MI) that utilize double-antibody/PEG technique [27]. Plasma leptin was also analyzed utilizing Linco RIA kits and was very similar to the plasma insulin method. 100 µL of plasma were incubated with 125I-labeled human leptin, normal rabbit IgG and rabbit anti-human leptin serum. After overnight incubation, a precipitating reagent containing goat anti-rabbit IgG serum was added; samples were mixed, and incubated for 20 min. Then, samples were centrifuged at 2500 x g for 20 minutes, liquid was decanted, and tubes containing the pellet were counted for 1 minute using a Cobra II-Auto Gamma Counting System.
Classification of Subjects with Insulin Resistance or the Metabolic Syndrome
Insulin resistance is defined as an impaired metabolic response to the body’s own insulin [28, 29] and is characterized by decreased capacity of insulin to promote typical glucose disposal. The homeostasis model assessment (HOMA) [30] was used to calculate insulin resistance according to the following equation: HOMA insulin resistance = fasting insulin (µU/mL) x fasting glucose (mmol/L) + 22.5. HOMA has been shown to be reliable for measuring insulin resistance in various populations when more invasive methods are not feasible [30]. Based on the equation, subjects were classified as having insulin resistance if calculated value was 
3.8 [31].
The subjects were classified as having the MS if 3 of the 5 risk factors delineated by the Adult Treatment Panel III (ATP III) were present: a fasting plasma glucose > 110 mg/dL (> 6.11 mmol/L), WC of > 88 cm, fasting TG > 150 mg/dL (> 1.70 mmol/L), fasting HDL-C of < 50 ( < 1.30 mmol/L) and blood pressure > 130 mm Hg (systolic) or 85 mm Hg (diastolic) [32].
Plasma Lipids
Our laboratory has participated in the Centers for Disease Control - National Heart, Lung and Blood Institute Lipid Standardization Program since 1989 for quality control and standardization for plasma TC, HDL-C and TG assays. Coefficients of variance during the study period were 0.76–1.42 for TC, 1.71–2.72 for HDL-C and 1.64–2.47 for TG. TC was determined enzymatically [33]. HDL-C was measured in the supernatant after precipitation of apo B-containing lipoproteins [34] and LDL-C was determined using the Friedewald equation [35]. TG were determined adjusting for free glycerol [36].
Urinary Carnitine
Urinary carnitine was determined by a spectrophotometric method. Briefly, 1 ml of perchloric acid was added to 1 ml of sample and mixed. After centrifugation at 5,000 x g for 10 min at 4° C, 200 µl of 1.75 M of potassium phosphate was added to the supernatant, mixed and centrifuged at 5,000 x g for 5 min at 4° C. 500 µl of the supernatant were then mixed with 2.5 µl of 5 M potassium hydroxide and incubated in a water bath for 15 min at 60° C. Following incubation, 120 µl of perchloric acid were added, mixed and centrifuged at 5,000 x g for 5 min at 4° C. From this mixture, 100 µl were mixed with 1 ml of 0.1 M Tris buffer and 100 µl of 1 mM DTNB. In a timed sequence, 5 µl of carnitine acetyltransferase and 30 µl of acetyl-CoA were added to this final mixture every 30 sec. Samples were incubated at RT for 15 min and read on a Spectrophotometer (Biomate 3, Thermo Spectronic, Rochester, NY) at 412 nm. Calculations were performed to determine concentrations of total acid soluble carnitine (TASC) in µM.
Statistical Analysis
Values are reported as mean ± standard deviation. Paired t tests compared changes in anthropometric, body composition measurements and glucose, insulin, and leptin concentrations between baseline and post-treatment. p < 0.05 was considered significant. Utilizing paired t-tests and Pearson correlations, no statistical significance was found due to L-carnitine supplementation in any of the measured parameters, therefore, data for all 70 subjects were pooled to evaluate the effects of weight loss (due to dietary modifications and increased physical activity) in all measured parameters. Data were analyzed using SPSS version 12.0 (SSPS, Chicago, IL). A non-parametric rank Wilcoxon test was used to evaluate the number of subjects having metabolic syndrome and insulin resistance at baseline and post-intervention. A stepwise linear regression was conducted to evaluate the major determinants of weight loss (increased physical activity, caloric reduction, increased intake of protein or decreased intake of carbohydrate). Similarly, changes in hormones were evaluated by a stepwise linear regression to determine the major determinants of the observed reductions in leptin and insulin. All data were analyzed using SPSS version 12.0 (SSPS, Chicago, IL).
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RESULTS |
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Baseline mean age, weight, and BMI were 29.4 ± 8.8 years, 79.4 ± 11.1 kg, and 29.6 ± 3.2 kg/m2, respectively. Mean systolic and diastolic blood pressures were 118.2 ± 7.3 mm Hg and 76.6 ± 6.9 mm Hg, respectively. There were no significant changes in blood pressure at 10 weeks. Participants had a mean decrease in energy intake of 2288.1 kJ (–26.6%) (p < 0.01) (Table 1).
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From baseline to 10 weeks, there was a significant increase (42.6%) of steps/day (3816, p < 0.01). TC, LDL-C, and TG decreased by 8%, 12.3%, and 19.5% (P < 0.01), respectively. Values were 4.7 ± 1.0, 2.6 ± 0.6, and 1.1 ± 0.5 mmol/L at baseline and 4.3 ± 0.7, 2.3 ± 0.6, and 0.7 ± 0.1 mmol/L after 10 weeks, respectively. In contrast, there were no changes in HDL-C from baseline to 10 weeks. Values were 1.57 ± 0.28 mmol/L at baseline and 1.59 ± 0.32 mmol/L post-intervention.
Body weight, BMI, and WC decreased by 4.6% (p < 0.01), 4.5% (p < 0.01), and 6.5% (p < 0.01), respectively (Table 2). Hip circumference and the WHR significantly decreased by 5% (p < 0.01) and 1.6% (p < 0.01), respectively. The intervention produced no significant changes in bone mineral density but did significantly change body composition measures (Table 3). Total body fat decreased by 4.0% (p < 0.001) and this corresponded to a decrease of 2.5 kg of fat mass (Table 3). Trunk fat also decreased by 4.7% (p < 0.001), a decrease of 1.3 kg of fat mass. Although there was a loss of lean tissue grams, the percent of lean tissue for whole body and trunk increased relative to fat mass by 3.0% and 3.4% respectively.
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DISCUSSION |
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The National Health and Nutrition Examination Surveys (NHANES) from 1971 to 2000 reported that females increased caloric intake by 21.7%, primarily due to increased carbohydrate consumption [40]. Simultaneously, lifestyles have become increasingly sedentary [41]. From NHANES III (1988–1994) to NHANES 1999–2000, females aged 20 to 29 years had an 8.7% increase in obesity and those aged 30 to 39 years experienced a 6.7% increase [42]. The largest increase (12.7%) was reported in women aged 60 to 69 years [42]. These statistics suggest that females do not modify behavior as they age and weight gain steadily continues. Strategies to promote weight loss, weight maintenance, and changes in the biological parameters to enhance both need to be elucidated.
Andersen et al compared structured aerobic exercise to increased lifestyle activity in overweight and obese adults [1, 5]. In one study, there was no significant difference between groups in relation to the amount of weight lost, loss of fat mass or fat free mass, or resting energy expenditure [1]. In the second study, there was no difference between groups in regards to improvements in weight, TC, and TG [5]. Therefore, increased physical activity could be promoted for weight loss and other health benefits for obese persons since both structured aerobic exercise and increased lifestyle activity promote similar benefits. In the current study, anthropometric and body composition data also support increasing lifestyle activities. The significant weight loss of the participants was mirrored by significant decreases in BMI and WC. WC has been shown to be a proxy measurement of abdominal obesity, especially visceral fat [43], and is associated with insulin resistance and dyslipidemia [44]. After controlling for BMI, WC significantly predicts CHD and DM2 [43]. A WC > 88 cm is considered high risk and 
88 cm is normal risk [21]. In this population, there was a mean decrease of 6.5% (P < 0.01) in WC from 90.1 ± 8.0 cm to 84.3 ± 7.8 cm, therefore making this group at lower risk. The decrease in WC was partly responsible for the significant decrease in incidence of MS seen in this study since a WC of > 88 cm is one of the five risk factors for this multifaceted syndrome. Elevated TG and decreased HDL-C are also risk factors for the MS. With this weight loss program, the mean TG concentration decreased and there was no significant change in mean HDL-C concentration. Other weight loss studies utilizing low-fat diets have resulted in decreased plasma HDL-C concentrations possibly due to decreased dietary fat intake [45]. HDL-C concentrations remained the same, possibly because our participants maintained dietary fat intake and increased their exercise.
The body composition changes also provide support for modest lifestyle modifications. Though there were significant decreases in grams of fat free mass, the overall percent of fat free mass increased. Some loss of lean tissue during weight loss is unavoidable, but maintaining as much lean tissue as possible is essential [46]. It is suggested that loss of lean tissue should not exceed 30% of total mass loss. In agreement with these recommendations, our population’s lean tissue loss accounted for 14.2% of total mass loss (lean body mass lost/total body mass lost * 100) and for 21.7% (trunk lean mass lost/total trunk mass lost * 100) of mass loss in the trunk.
Although no significant decrease in plasma glucose concentrations occurred, insulin and leptin concentrations significantly decreased by 17.9% and 5.9%, respectively. Insulin activity has been shown to decrease in the obese state, due to a decrease in lipolysis activation [47]. Furthermore, decreased body fat has shown to increase lipolysis efficiency [10]. Volek et al [48] explored the effects of a very low carbohydrate diet on body composition and insulin response and postulated that the observed decreases in insulin concentrations may have influenced both weight and fat mass loss. Similar effects were observed in the current study. The decreased carbohydrate intake with increased protein intake reduced concentrations of insulin, thereby contributing to the overall weight loss and reduction in fat mass. Leptin concentrations are higher in females than in males, which may affect leptin transport into the central nervous system [12]. Therefore, larger reductions in leptin need to be experienced by females to effect energy balance [12]. The decrease in insulin concentrations may have been responsible for the significant decrease in number of subjects with insulin resistance. Because unmanaged insulin resistance can result in DM2, weight loss strategies, such as the one utilized in this study, need to be further researched.
In conclusion, an intervention consisting of a moderate hypocaloric diet with modified macronutrient composition, in addition to increased daily physical activity, successfully impacted multiple risk factors for DM2 and CHD. The current weight loss intervention beneficially changed body composition, plasma lipids, and insulin and leptin concentrations. Most importantly, the number of subjects classified with insulin resistance and the metabolic syndrome were substantially reduced. This study suggests that moderate lifestyle changes, if followed and maintained, can decrease the risk for chronic disease.
Received May 17, 2005. Accepted October 18, 2005.
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REFERENCES |
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