Consumption of High-Fructose Corn Syrup Compared with Sucrose Promotes Adiposity and Increased Triglyceridemia but Comparable NAFLD Severity in Juvenile Iberian Pigs

Abstract

Background: Fructose consumption has been linked to nonalcoholic fatty liver disease (NAFLD) in children. However, the effect of high-fructose corn syrup (HFCS) compared with sucrose in pediatric NAFLD has not been investigated.

Objectives: We tested whether the isocaloric substitution of dietary sucrose by HFCS would increase the severity of NAFLD in juvenile pigs, and whether this effect would be associated with changes in gut histology, SCFA production, and microbial diversity.

Methods: Iberian pigs, 53-d-old and pair-housed in pens balanced for weight and sex, were randomly assigned to receive a mash diet top-dressed with increasing amounts of sucrose (SUC; n = 3 pens; 281.6-486.8 g/kg diet) or HFCS (n = 4; 444.3-724.8 g/kg diet) during 16 wk. Diets exceeded the animal's energy requirements by providing sugars in excess, but met the requirements for all other nutrients. Animals were killed at 165 d of age after blood sampling, and liver, muscle, and gut were collected for histology, metabolome, and microbiome analyses. Data were analyzed by multivariate and univariate statistics.

Results: Compared with SUC, HFCS increased subcutaneous fat, triacylglycerides in plasma, and butyrate in colon (P ≤ 0.05). In addition, HFCS decreased UMP and short-chain acyl carnitines in liver, and urea nitrogen and creatinine in serum (P ≤ 0.05). Microbiome analysis showed a 24.8% average dissimilarity between HFCS and SUC associated with changes in SCFA-producing bacteria. Body weight gain, intramuscular fat, histological and serum markers of liver injury, and circulating hormones, glucose, and proinflammatory cytokines did not differ between diets.

Conclusions: Fructose consumption derived from HFCS promoted butyrate synthesis, triglyceridemia, and subcutaneous lipid deposition in juvenile Iberian pigs, but did not increase serum and histological markers of NAFLD compared with a sucrose-enriched diet. Longer studies could be needed to observe differences in liver injury among sugar types.

Keywords: added sugar; dysbiosis; metabolomics; microbiome; pediatric.

FIGURE 1

Hypercaloric diet supplemented with a high-fructose corn syrup (HFCS) decreased lean mass composition in juvenile Iberian pigs compared with a sucrose-enriched diet (SUC). (A) Cumulative body weight gain in baseline (BSL, n = 3 pens), SUC (n = 3 pens), and HFCS (n = 4 pens) pigs was measured every 7 d during 16 wk. (B) Lean mass was calculated using the formula 100 × [8.588 + (0.465 × hot carcass weight) − (21.896 × 10th rib fat depth) + (3.005 × 10th rib loin muscle area)]/hot carcass weight. (C) Dorsal fat was measured at the 10th rib in both sides of the pig loins. Values are means ± SDs. Data were analyzed using 1-factor ANOVA. Labeled means without a common letter differ.

FIGURE 2

Histological markers of liver injury did not differ between juvenile Iberian pigs fed hypercaloric diets enriched with either sucrose (SUC) or high-fructose corn syrup (HFCS). (A) Colorimetric detection of total glycerides cleaved from monoacylglycerides, diacylglycerides, and triacylglycerides in liver tissue of baseline (BSL; n = 3 pens), SUC (n = 3 pens), and HFCS (n = 4 pens) pigs, expressed as millimolar per gram of tissue. (B) Representative microscopy images of liver tissue stained with hematoxylin-eosin (H&E) and trichrome methods (scale bars: 50 μm in H&E staining and 200 μm in trichrome slides). Steatosis, ballooning degeneration, Mallory–Denk bodies, lobular inflammation, fibrosis, and necrosis were semiquantitatively evaluated for each pig, and a lesion score was calculated. The zonal distribution of each of these variables was also annotated. (C) Percentage of Iberian pigs with histopathological changes in liver tissue consistent with steatosis based on the above described semiquantitative histological evaluation of all the pig livers. Values are means ± SDs. Data were analyzed using 1-factor ANOVA.

FIGURE 3

High-fructose corn syrup (HFCS)-enriched diet decreased UMP and short-chain acyl carnitines in liver, and increased triacylglycerides in plasma. Principal coordinate analysis of liver (A) and plasma (B) metabolites in juvenile Iberian pigs fed baseline (BSL; n = 3 pens), sucrose (SUC; n = 3 pens), or HFCS (n = 4 pens) diets to visualize group discrimination in a 2-dimensional scores plot. Each point represents an individual pig and color of point denotes diet. Group differences were assessed by nonparametric permutational ANOVA with diet as fixed effects, and pen nested in diet as random effect, under a reduced model, 9999 permutations, and type III sum of squares. (C) Heatmap of metabolites significantly altered by the diet in liver and blood, with fold change and significance levels by HFCS compared with SUC. Metabolites were measured by ultra-performance LC tandem quadrupole MS. Columns are individual pigs and rows are log₂-transformed metabolites. Blue and red colors represent the row minimum and maximum values. Identification of significant metabolites was performed by 1-factor ANOVA and a mixed model that included diet as fixed effect, and pen nested in diet as random effect. Significance level for each metabolite was corrected for the total number of tests using the Benjamini–Hochberg (FDR) procedure. Using a raw ANOVA P value ≤ 0.05, we identified 4 and 9 significant metabolites in liver and plasma, respectively, whereas using an FDR-corrected ANOVA P value ≤ 0.05, only 1 metabolite (sucrose + maltose + lactose) differed between diets. ^*,**,***Significant fold change for HFCS compared with SUC: *P ≤ 0.05, **P ≤ 0.01, ***P  ≤ 0.001. FDR, false discovery rate; PCoA, principal coordinate analysis; PE, phosphatidylethanolamine; SM, sphingomyelin; , TAG, triacylglyceride.

FIGURE 4

Inclusion of high-fructose corn syrup (HFCS) in a hypercaloric diet increased colonic concentrations of butyric acid in juvenile Iberian pigs compared with a sucrose-enriched diet (SUC). (A) Heatmap of SCFAs in colon digesta of baseline (BSL; n = 3 pens), SUC (n = 3 pens), and HFCS (n = 4 pens) pigs quantified by GC, with fold change and significance levels by HFCS compared with SUC (*P ≤ 0.05). Heatmap columns are individual pigs and rows are log₂-transformed metabolites. Blue and red colors represent the row minimum and maximum values. Identification of significant SCFAs was performed by 1-factor ANOVA and a mixed model that included diet as fixed effect, and pen nested in diet as random effect. (B) Representative microscopy images of distal ileum and colon samples from juvenile Iberian pig stained with hematoxylin-eosin (H&E), showing measurements of villi length and crypt depth in the mucosa (scale bars, 200 μm). H&E stains were examined using an optical microscope (Axioscop 40; Zeiss) fitted with a camera (AxioCam MRc; Zeiss) and the images were analyzed using image analysis software (Axiovision SE64 4.7.1; Zeiss). Four fields of view per sample were captured with a final magnification of 40×, and the height and depth of every well-oriented villus and crypt, respectively, were measured in micrometers and used to calculate an average value for each intestinal segment of each pig. A villus was deemed well oriented if it was visible from the apex of the villus to the base, whereas well-oriented crypts were adjacent to villi and visible from the luminal opening to the bottom of the crypt. (C) Quantitative analysis of villus length and crypt depth in distal ileum and colon samples. Data were analyzed using 1-factor ANOVA and presented as means ± SDs.

FIGURE 5

Microbiome dissimilarity between high-fructose corn syrup (HFCS) and sucrose (SUC) colon samples was associated with changes in SCFA-producing bacteria. (A) Principal coordinate analysis of Bray–Curtis dissimilarity matrix on taxa at the genus level obtained by 16S rRNA gene amplification of the V4 region in colon content samples from baseline (BSL; n = 3 pens), SUC (n = 3 pens), and HFCS (n = 4 pens). Each point represents an individual pig and color of point denotes diet. Permutational ANOVA results including P value, R², and F-statistic were calculated using the compare_categories.py script available in QIIME. (B) Observed OTUs, Shannon and Chao1 indices of α diversity at the genus level were computed using the core_diversity_analyses.py script, with a rarefaction of 98,767 counts/sample. (C) Cumulative percentage contribution of genera to dissimilarity between HFCS and SUC (left y axis), and their corresponding ratio of average dissimilarity to its SD (right y axis) were computed using a similarity percentage analysis with 80% cut-off value. OTU, operational taxonomic unit; PCoA, principal coordinate analysis; Phascolarctobact, Phascolarctobacterium; QIIME, Quantitative Insights Into Microbial Ecology.