Abstract
High-fructose diet stimulates hepatic de novo lipogenesis (DNL) and causes hypertriglyceridemia and insulin resistance in rodents. Fructose-induced insulin resistance may be secondary to alterations of lipid metabolism. In contrast, fish oil supplementation decreases triglycerides and may improve insulin resistance. Therefore, we studied the effect of high-fructose diet and fish oil on DNL and VLDL triglycerides and their impact on insulin resistance. Seven normal men were studied on four occasions: after fish oil (7.2 g/day) for 28 days; a 6-day high-fructose diet (corresponding to an extra 25% of total calories); fish oil plus high-fructose diet; and control conditions. Following each condition, fasting fractional DNL and endogenous glucose production (EGP) were evaluated using [1-13C]sodium acetate and 6,6-2H2 glucose and a two-step hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity. High-fructose diet significantly increased fasting glycemia (7 ±2%), triglycerides (79 ± 22%), fractional DNL (sixfold), and EGP (14 ± 3%, all P < 0.05). It also impaired insulin-induced suppression of adipose tissue lipolysis and EGP (P < 0.05) but had no effect on whole- body insulin-mediated glucose disposal. Fish oil significantly decreased triglycerides (37%, P < 0.05) after high-fructose diet compared with high-fructose diet without fish oil and tended to reduce DNL but had no other significant effect. In conclusion, high-fructose diet induced dyslipidemia and hepatic and adipose tissue insulin resistance. Fish oil reversed dyslipidemia but not insulin resistance.
Over the past decades, per capita consumption of high-fructose corn syrup has increased dramatically. Several authors suggest that increased fructose ingestion may be responsible for the present epidemic of obesity and the increased incidence of metabolic syndrome and diabetes (1). Diets rich in simple sugars, particularly fructose, have been shown to be associated with hypertriglyceridemia both in humans (2) and rodents (3). This may be due to stimulation of hepatic de novo lipogenesis (DNL) and increased secretion of triglyceride-rich particles by the liver or to decreased extrahepatic clearance of triglyceride particles (4,5). Moreover, there is evidence that high-fructose diets can lead to insulin resistance in rodents (3,6). To further delineate the metabolic consequences of fructose overconsumption, we measured fractional hepatic DNL and insulin sensitivity in the liver, adipose tissue, and at the whole- body level. This was performed in a group of healthy male volunteers after 6 days of either fructose overfeeding or an isoenergetic, low-fructose diet. Since n-3 polyunsaturated fatty acids are known to prevent hypertriglyceridemia and the development of insulin resistance in dietary models of obesity in rats (6) and may suppress hepatic lipogenic enzymes (7), each participant was also studied after a 4-week fish oil supplement.
RESEARCH DESIGN AND METHODS
Seven healthy male volunteers without family history of diabetes were recruited by advertisement. They were aged 22–31 years and had BMIs of 20.2–25.4 kg/m2 (Table 1). All subjects were in apparent good health, were nonsmokers, and took no medications. The study was approved by the ethical committee of Lausanne University School of Medicine and a written consent was obtained from each subject after the nature of the study was explained.
Anthropometry and body composition measurements.
Standing height was measured using a stadiometer. Body weight and hip and waist circumferences were measured before the last meal preceding each study (8). Body composition was estimated from subcutaneous skin fold thickness measurements at the biceps, triceps, subscapular, and suprailiac sites as described by Durnin and Womersley (9).
Study design.
Each subject was studied on four occasions (Fig. 1). On one occasion, volunteers received 7.2 g of fish oil (1.2 g eicosapentaenoic acid and 0.8 g docasahexaenoic acid; Biorganic Omega-3, Gisand, Bern, Switzerland) per day for 28 days. It has been documented that such supplementation with fish oils leads to marked increases in n-3 fatty acids in serum phospholipids (10,11). On another occasion, subjects ingested 3 g of fructose (D-Fructose; Fluka Chemie Gmbh, Buchs, Switzerland) per kilogram of body weight per day (high-fructose diet) as a 20% fructose solution with the three main meals during the 6 days before the test. On the third occasion, fish oil supplementation was combined with high-fructose diet. Each subject also underwent a control test. Volunteers were instructed to avoid certain foods and to follow a balanced, isoenergetic diet, which was controlled by a food diary during an initial 3-day period. Thereafter, during the 3 days preceding each test, the subject followed a provided isoenergetic diet (15% proteins, 35% lipids, 40% starch, 10% mono- and disaccharides) partitioned into three meals at 0700, 1200, and 1900 and two snacks at 0900 and 1500. During the high-fructose diet, subjects received the same diet supplemented with 3 g · kg−1 · day−1fructose, resulting in an hyperenergetic (800–1,000 kcal/day) diet containing 11% proteins, 26% lipids, 30% starch, 8% glucose and disaccharides, and 25% fructose. Subjects were told to avoid vigorous physical activity during the 6 days preceding the tests.
The order by which the four dietary conditions were applied was randomized, with an interval of 12 weeks after the two tests with fish oil administration to allow wash-out of fish oil between the experiments.
Fasting hepatic DNL and endogenous glucose production.
After each dietary condition, subjects underwent an overnight 13-h study (from 2200 to 1100 of the next morning), during which they stayed in bed and slept between 2200 and 0600. On the evening of the study, they took their last meal at 1830. At 2030, two indwelling catheters were inserted: one into a right wrist vein for blood sampling; the other into a vein of the controlateral forearm for infusions. From 2200 to 0730, 0.5 g/h of [1-13C]sodium acetate (10 mg/ml in NaCl 0.9%) was infused constantly. Whole-body glucose turnover was assessed with 6,6-2H2 glucose infusion (bolus: 2 mg/kg; continuous: 20 μg · kg−1 · min−1) between 0500 and 1100. Basal blood samples were obtained at 0600, 0700, and 0730 for determination of hepatic DNL, endogenous glucose production (EGP), insulin, glucose, nonesterified fatty acid (NEFA), and triglyceride concentrations (referred as “fasting condition”).
Hyperinsulinemic-euglycemic clamp.
To assess insulin sensitivity, insulin-mediated glucose disposal (6,6-2H2glucose, “hot infusion model”) (12), inhibition of EGP, and suppression of lipolysis (plasma NEFA concentrations) and lipid oxidation (indirect calorimetry), a two-step (0.2 mU · kg−1 · min−1 from t = 0 to t = 90, then 0.5 mU · kg−1 · min−1 from t = 90 to t = 180) hyperinsulinemic-euglycemic clamp (13) was performed between 0800 and 1100 (Fig. 1). Blood samples were collected at 30-min intervals to measure insulin, NEFA, and triglycerides concentrations. The glucose infusion rate (insulin resistance) during the clamp was used to evaluate insulin-mediated glucose disposal. Indirect calorimetry was performed using a ventilated canopy as described previously (14) from 0700 until 1100 (Fig. 1). Energy expenditure and substrate oxidation rates were measured using the equation of Livesey and Elia (15).
Analytical procedures.
Blood samples were immediately centrifuged at 4°C to separate plasma, which was then frozen at −20°C until testing. Plasma glucose and lactate concentrations were measured with a glucose-lactate analyzer YSI 2300 STAT Plus (Yellow Springs, OH). Plasma NEFA and triglycerides concentrations were analyzed by a colorimetric method using commercial kits for NEFA (NEFA C; Wako Chemicals, Freiburg, Germany) and for triglycerides (Biomérieux Vitek, Switzerland). Commercial radioimmunoassay and enzyme-linked immunosorbent assay kits were used for determination of plasma insulin (Biochem Immunosystems, Freiburg, Germany), adiponectin (Linco Research, St. Charles, MO), and resistin (Human Resistin ELISA, BioVendor Laboratory Medicine, Czech Republic). During the clamp, plasma glucose concentrations were measured by the glucose oxidase method using a Beckman glucose analyzer II (Beckman Instruments, Fullerton, CA). Plasma 6,6- 2H2glucose isotopic enrichment was measured by gas chromatography–mass spectrometry (Model 5973; Hewlett-Packard, Palo Alto, CA) as described (16). Plasma VLDL-13C palmitate enrichment and mass isotopomers were measured as described by Hellerstein et al. (17). Gas chromatography–mass spectrometry was used for analysis of isotopic enrichments of plasma fatty acid-methyl esters from VLDL. For fatty acid-methyl esters analysis, a 25-m fused DB-1 silica column was used, with electron impact ionization ion at m/z 270–272 representing the parent M0 through the M2 isotopomers (17). Fractional DNL was calculated by the isotopomer distribution analysis technique. The ratio of excess double-labeled to excess of single-labeled species (EM2/EM1) in VLDL palmitate reveals the isotope enrichment of the true precursor pool for lipogenesis (hepatic cytosolic acetyl-CoA) by application of probability logic based on the multinomial expansion. The fractional contribution from DNL to VLDL fatty acid can then be calculated by the precursor-product relationship (17).
Statistical analysis.
Data are expressed as means ± SE. Statistical analyses were performed by using STATA version 8.2 (StataCorp, College Station, TX) with P < 0.05 as level of significance. Global mean difference among the various conditions was tested using the Friedman’s test. When a significant difference was found, multiple comparisons between two conditions were performed using the paired Wilcoxon test. To test the existence of a trend, we performed the Page’s test (18).
