Saturated and Unsaturated Dietary Fats Differentially Modulate Ethanol-Induced Changes in Gut Microbiome and Metabolome in a Mouse Model of Alcoholic Liver Disease

Abstract

Alcoholic liver disease (ALD) ranks among major causes of morbidity and mortality. Diet and crosstalk between the gut and liver are important determinants of ALD. We evaluated the effects of different types of dietary fat and ethanol on the gut microbiota composition and metabolic activity and the effect of these changes on liver injury in ALD. Compared with ethanol and a saturated fat diet (medium chain triglycerides enriched), an unsaturated fat diet (corn oil enriched) exacerbated ethanol-induced endotoxemia, liver steatosis, and injury. Major alterations in gut microbiota, including a reduction in Bacteroidetes and an increase in Proteobacteria and Actinobacteria, were seen in animals fed an unsaturated fat diet and ethanol but not a saturated fat diet and ethanol. Compared with a saturated fat diet and ethanol, an unsaturated fat diet and ethanol caused major fecal metabolomic changes. Moreover, a decrease in certain fecal amino acids was noted in both alcohol-fed groups. These data support an important role of dietary lipids in ALD pathogenesis and provide insight into mechanisms of ALD development. A diet enriched in unsaturated fats enhanced alcohol-induced liver injury and caused major fecal metagenomic and metabolomic changes that may play an etiologic role in observed liver injury. Dietary lipids can potentially serve as inexpensive interventions for the prevention and treatment of ALD.

Figure 1

The experimental animal model of alcoholic liver disease. A: The schematic presentation of chronic ethanol (EtOH) or control diet feeding protocol. Initially, all mice were given the control liquid maltose dextrin diets [saturated fat (SF) or unsaturated fat (USF), no ethanol] ad libitum for 1 week. Afterward, mice were fed either the liquid ethanol-containing diets or the control liquid maltose-dextrin diets. Ethanol was gradually increased to 35% of total calories (5.0% v/v). The mice were fed the ethanol diet (5% ethanol v/v) ad libitum for 8 weeks. B: The composition of the experimental liquid diets. The SF diet was enriched with medium chain triglyceride oil and beef tallow fat (82:18 ratio). The USF diet was enriched with corn oil. Soybean oil was used in both diets to provide essential free fatty acids. The control (SF and USF) diets contained 43% of calories from carbohydrate, 17% from protein, and 40% from fat. The SF and ethanol and USF and ethanol diets contained 35% of calories from ethanol to replace the calories from carbohydrate.

Figure 2

Effects of different dietary fat profile on liver steatosis, injury, and endotoxemia in response to chronic alcohol feeding. A: Representative images of hepatic hematoxylin and eosin (H&E) staining. B: Liver triglyceride (TG) levels. C: Plasma alanine aminotransferase (ALT) levels. D: Plasma lipopolysaccharide (LPS) levels. Data are expressed as means ± SEM. n = 6 to 7 animals per group. ^∗P < 0.05, two-way analysis of variance, followed by the Tukey multiple-comparison test. Original magnification, ×200 (A). EtOH, ethanol; SF, saturated fat; USF, unsaturated fat.

Figure 3

Effects of different types of dietary lipids and chronic ethanol (EtOH) administration on hepatic apoptosis and neutrophil and macrophage infiltration. A and B: Representative images and quantitation of chloroacetate esterase (CAE) staining. Arrows indicate CAE-positive neutrophils. C and D: Representative images and quantitation of F4/80 staining. The brown staining represents F4/80-positive hepatic macrophages. E and F: Representative images of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Arrows indicate TUNEL-positive hepatocytes. Quantitation of representative images was performed by blinded counting of the number of CAE-, F4/80-, and TUNEL-positive cells in random 7 to 10 digital images per animal (I.A.K. and J.I.B.). Data are expressed as means ± SEM. n = 3 animals per group. ^∗P < 0.05. Original magnification: ×200 (A, C, and E). SF, saturated fat; USF, unsaturated fat.

Figure 4

Effects of different types of dietary lipids and chronic ethanol (EtOH) administration on gut microbiota composition. A: Comparative analysis of the gut bacteria relative abundance in mice fed ethanol and different types of dietary lipids for 8 weeks. The phyla abundance is indicated by the color bars. Uncommon phyla that are a very small fraction of the total may not be visible in the chart even though they are present in the legend. B: Firmicutes:Bacteroidetes ratio at the end of the experiment. C: Changes in the gut bacteria phylum occur in response to 8 weeks of ethanol feeding compared with baseline. Results are presented as fold-changes over baseline set as 0. SF, saturated fat; USF, unsaturated fat.

Figure 5

Effects of dietary fat, ethanol (EtOH), and their combination on changes of fecal free fatty acids after 8 weeks of experiment. A: Heptadecanoic (margaric, C₁₇H₃₄O₂) acid. B: 9-cis-Hexadecenoic (palmitoleic, C₁₆H₃₀O₂) acid. C: Octanoic (caprylic, C₈H₁₆O₂) acid. D: Hexanoic (caproic, C₆H₁₂O₂) acid. E: Butanoic (butyric, C₄H₈O₂) acid. Results are presented as fold-changes over the saturated fat (SF) set as 1. ^∗P < 0.05. USF, unsaturated fat.