Participant recruitment and screening

Participants were recruited through posted flyers, emails through Listservs, word of mouth, StudySearch, and by using ResearchMatch through The Ohio State University Center for Clinical and Translational Science. Print and email advertisements instructed interested individuals to call the study center, and one of the key personnel involved in the project described the study and determined preliminary qualifications by conducting a scripted phone interview. Participant answers to qualifying criteria questions were recorded to assess whether or not the person calling met the initial qualifying criteria. If participants met the initial qualifying criteria, an in-person appointment was scheduled.

Inclusion criteria included men and women 21 to 65 years of age with MetS defined as having 3 or more of the following characteristics: (a) waist circumference > 102 cm (40 in) in men and 88 cm (35 in) in women; (b) triglycerides ≥ 150 mg/dL; (c) HDL-C < 40 mg/dL in men, and < 50 mg/dL in women; (d) BP ≥ 130/85 mmHg; and (e) fasting glucose ≥ 100 mg/dL (47). Potential participants were excluded who had high total cholesterol (> 300 mg/dL), diabetes, liver, kidney, or other metabolic or endocrine dysfunction, gastrointestinal disorders, smoked regularly, had lactose intolerance, consumed excessive alcohol, or used medications for high cholesterol, diabetes, or infections in the last 3 months.

During the screening meeting, interested participants provided informed consent in accordance with the Institutional Review Board at The Ohio State University and the Declaration of Helsinki. After consent was provided, anthropometric measures of height, body mass, waist circumference, and BP were obtained. Additionally, a venipuncture in the antecubital fossa was conducted in the fasted state. A screening chemistry panel including serum lipids and glucose was sent to Quest Diagnostics for analysis. Participants were also required to complete an MRI screening questionnaire to determine if the participant could safely have an MRI scan completed. A flowchart of participant recruitment and enrollment can be found in Supplemental Figure 2. A total of 16 participants with MetS were enrolled: 7 met 3 out of 5 criteria, 6 met 4 out of 5 criteria, and 3 met all 5 criteria.

Experimental approach

Information about the sample size and baseline characteristics can be found in the Results section and in Figure 2. After enrollment, participants completed 3 controlled-feeding periods each lasting 4 weeks with a 2-week washout period in between diets (Figure 1A). The order of diets was randomized and balanced. During a 2-week run-in period, participants were fed an MC, standard American diet to determine an appropriate energy expenditure to maintain body mass. All controlled diets were formulated to consist of the same total calories to maintain weight stability throughout the entire experimental period. Testing occurred at baseline and was repeated after each of the controlled diets.

Method details

Controlled feeding and dietary intervention formulation. Specific 7-day rotational menus were developed for the 3 diet treatments: LC, MC, and HC using typical food items. Each menu was designed using a caloric intake base of 2,500 kcal and to allow for scaling options for various caloric intakes. Although individual differences in absolute intakes of food occurred, this approach allowed for relative macronutrient and micronutrient proportions to be constant between participants.

Participants’ caloric needs were preliminarily determined via indirect calorimetry and the Harris-Benedict equation. At the initiation of the run-in diet, participants were fed an MC diet at a caloric intake level estimated to match energy expenditure. Any changes in body mass were monitored and caloric intake adjusted accordingly to maintain weight stability. Once body mass stabilized, no further adjustments to caloric intake were made across the interventions.

All food was prepared and provided to subjects during the experimental period. All food, drinks, and seasonings were weighed to the nearest 0.1 g and prepared in a metabolic kitchen located at The Ohio State University. In order to minimize intercompany and interproduct variations, every product utilized in the trial was maintained throughout the entirety of the study. All food was prepared by baking, boiling, or sautéing and all juices were collected in order to minimize nutrient loss. Participants were instructed to eat/drink meals in their entirety, including consumption of any residual oils that may be in the containers. Adherence was tracked by the receipt of empty food containers. Detailed nutrient composition was completed a priori via Nutritionist Pro (Axxya Systems) for every meal to ensure accurate macro-/micronutrient composition. Mean nutrient intakes for the diet periods are reported in Supplemental Table 2.

All 3 investigational diets were isocaloric, isonitrogenous, and contained a scalable amount of cheese (Cheddar and Gouda) that approximated 6 oz/day of full-fat cheese per 2,500 kcal. The MC diet was created to be comparable to the standard American diet with approximately one-third energy from fat and half from carbohydrate. It was high in potatoes and a mix of whole and processed grains, with at least 5 servings of fruits and vegetables every day. For the HC diet, we cut out fat primarily from animal products (except cheese) and scaled carbohydrate proportionately with at least 5 servings of fruits and vegetables per day. For the LC diet, we did the opposite. Because the primary vector for saturated fat was provided in the form of full-fat cheese products, polyunsaturated fat was relatively low for all diets. The main sources of polyunsaturated fat came from fatty meats, nuts, and condiments such as mayonnaise.

Metabolic analysis

Substrate oxidation rates, respiratory exchange ratio, and resting energy expenditure were measured via indirect calorimetry (Parvomedics TrueOne 2400) in the early morning after an overnight fast. After participants’ arrival and hydration status assessment, they were brought into a dark, quiet, room (ambient temperature: 20°C–22°C) to relax for 30 minutes. During this time and throughout the duration of the assessment, participants were supine on an examination table with pillows under their head and knees to ensure comfort throughout the testing period. Prior to subject assessment, the metabolic carts were calibrated with standardized gas and pressure. Participants laid motionless and were not allowed to talk or fall asleep during the assessment. Both expired volume of carbon dioxide and inspired oxygen were collected and sampled for a period of 40 minutes. The first 10 minutes of sampling was utilized as a stabilization period to account for any breathing alterations that may occur at the onset of assessment. As such, the total sampling time for determining substrate oxidation and resting energy expenditure was 30 minutes.

Venipuncture and blood chemistry

All blood was collected by a trained phlebotomist via venipuncture in the morning after an overnight fast. All venous blood was collected from the antecubital fossa via 21-g or 23-g butterfly needles (BD Vacutainer Safety-Lok Blood Collection Set, Becton, Dickinson and Company). Blood was collected into 10-ml spray-coated K2EDTA, sodium heparin, serum, and serum separator tubes (BD Vacutainer). Plasma tubes were placed on ice, while serum tubes were allowed to sit at room temperature until clotted (30–45 minutes). Blood collection tubes were then centrifuged at 1,200 g for 10 minutes. The serum separator tubes were stored at 4°C until courier service collected daily and were analyzed by Quest Diagnostics for serum glucose, triglycerides, LDL, and HDL. Remaining serum and plasma was then aliquoted and snap-frozen in liquid nitrogen. All samples were stored at –80°C and allowed only 1 free-thaw cycle before assay. Serum insulin was determined via ELISA with an average coefficient of variation of 2.1% (Quantikine, catalog no. DINS00, R&D Systems). Insulin resistance was determined from fasting glucose and insulin (48).

Body composition and anthropometric analysis

Whole-body composition and bone mineral density analysis was completed using an iDXA (GE Healthcare). All measurements were conducted by a radiologist certified in this technology. Bone mineral density, total lean mass, and fat mass were calculated. All waist circumference measurements were conducted by a trained researcher in the area between the floating ribs and the iliac crest, as defined by the WHO (Gulick II, Fitness Mart). Height and fasting body mass were assessed in the laboratory using a stadiometer and scale (SECA Model 703). BP was determined using a BP cuff (Welch Allyn) by a trained researcher after participants had been seated quietly, with feet on the floor, for a period of 5 minutes. Two measurements were taken with 2 to 3 minutes between subsequent measures.

MRI-based fat quantification

We assessed visceral adipose tissue (VAT) mass and liver organ fat fraction by MRI using a 3-Tesla system (MAGNETOM, Tim Trio, Siemens Healthcare). The abdomen, including the entire liver, was scanned during a single breath hold with a slice thickness of 5 mm. Fat percentage and water percentage image maps were generated automatically using the variable projection (VARPRO) fat separation technique (49). The resulting digital imaging and communications in medicine images were processed using semiautomated, custom-built software to segment VAT and subcutaneous adipose tissue (SAT) compartments and calculate the volume and mass of fat within the abdominal VAT and SAT depots.

Hepatic fat percentage was quantified using the same VARPRO image data used for VAT measurements. This technique provides an accurate and objective measurement of tissue fat composition on a pixel-by-pixel basis. Using the fat percentage maps automatically generated by the VARPRO technique, fat fraction was measured in regions of interest manually placed in each of the 9 standard anatomical liver segments (50). The regions of interest were drawn to avoid veins and visible image artifacts. Three measurements were taken within each segment (in 3 different slice planes) for a total of 27 measurements. The hepatic fat fraction (%) was expressed as the average fat fraction across all 9 liver segments.

Plasma fatty acid profiling of PL and TG

Plasma samples were extracted with mixtures of chloroform and methanol and extracted by standard methods of Bligh and Dyer (51). Prior to extraction, 100 μg phosphatidyl choline and triglyceride containing 17:0 (heptadecanoic acid) acyl chains was added as an internal standard. Resulting lipid extracts were dried under a steady stream of nitrogen. Dried lipid extracts were reconstituted in 100 μL chloroform and spotted on silica gel thin layer chromatography plates and developed in hexane/diethyl ether/glacial acetic acid (80:20:1). Resulting lipid bands were visualized with 5% dichlorofluorescein (Sigma-Aldrich) with ultraviolet light. The bands corresponding to total polar lipids (phospholipids) and triglycerides were scraped into a 16- × 100-mm screw-top Pyrex test tube. The lipid classes were then transesterified with 2% sulfuric acid in methanol in a hot water bath at 75°C. The resulting fatty acid methyl esters were then extracted with the addition of water and petroleum ether. The fatty acid methyl esters were then analyzed with a Shimadzu 2010 gas chromatograph (Shimadzu Corporation) utilizing a FAMEWAX capillary column (Restek) and flame ionization detector. Relative amounts of fatty acids in each lipid class were determined with internal standards and fatty acids from 12 to 24 carbons were quantified. Fatty acid data are expressed as both weight and molar percent composition.

Plasma lipoprotein particle analysis

Particle concentrations of VLDL, intermediate-density lipoprotein (IDL), LDL, and HDL subfractions were analyzed in specific particle-size intervals using ion mobility, which uniquely allows for direct particle quantification as a function of particle diameter (52) following a procedure to remove other plasma proteins (53). The ion mobility instrument utilizes an electrospray to create an aerosol of particles, which then pass through a differential mobility analyzer coupled to a particle counter. Particle concentrations (nmol/l) are determined for subfractions defined by the following size intervals (nm): VLDL: large (42.40–54.70), medium (33.50–42.39), small (29.60–33.49); IDL: large (25.00–29.59), small (23.33–24.99); LDL: large (22.0–23.32), medium (21.41–21.99), small (20.82–21.40), very small (18.0–20.81); HDL: large (10.50–14.50) and small (7.65–10.49). Peak LDL diameter (nm) is determined as described by Caulfield et al. (52). Interassay variation was reduced by inclusion of 2 in-house controls in each preparatory process and triplicate analysis. CV < 15% for each subfraction measurement was maintained throughout.

Statistics

A repeated-measures ANOVA was performed to test for differences in the various outcome variables after the 3 diet periods (i.e., LC, MC, and HC). In a few cases, data that did not have a normal distribution was log transformed. Main effects were evaluated by Fisher’s least significant difference post hoc testing. A significance level of P ≤ 0.05 was chosen. Statistical analyses were performed using Statistica.

Study approval

Prior to participation, all subjects in this study signed an informed consent document approved by the Institutional Review Board at The Ohio State University.