Combination of alcohol and fructose exacerbates metabolic imbalance in terms of hepatic damage, dyslipidemia, and insulin resistance in rats

Abstract

Although both alcohol and fructose are particularly steatogenic, their long-term effect in the development of a metabolic syndrome has not been studied in vivo. Consumption of fructose generally leads to obesity, whereas ethanol can induce liver damage in the absence of overweight. Here, Sprague-Dawley rats were fed ad libitum for 28 days on five diets: chow (control), liquid Lieber-DeCarli (LDC) diet, LDC +30%J of ethanol (L-Et) or fructose (L-Fr), and LDC combined with 30%J ethanol and 30%J fructose (L-EF). Body weight (BW) and liver weight (LW) were measured. Blood and liver samples were harvested and subjected to biochemical tests, histopathological examinations, and RT-PCR. Alcohol-containing diets substantially reduced the food intake and BW (≤3rd week), whereas fructose-fed animals had higher LW than controls (P<0.05). Additionally, leukocytes, plasma AST and leptin levels were the highest in the fructose-administered rats. Compared to the chow and LDC diets, the L-EF diet significantly elevated blood glucose, insulin, and total-cholesterol levels (also vs. the L-Et group). The albumin and Quick-test levels were the lowest, whereas ALT activity was the highest in the L-EF group. Moreover, the L-EF diet aggravated plasma triglyceride and reduced HDL-cholesterol levels more than 2.7-fold compared to the sum of the effects of the L-Et and L-Fr diets. The decreased hepatic insulin clearance in the L-EF group vs. control and LDC groups was reflected by a significantly decreased C-peptide:insulin ratio. All diets except the control caused hepatosteatosis, as evidenced by Nile red and H&E staining. Hepatic transcription of insulin receptor substrate-1/2 was mainly suppressed by the L-Fr and L-EF diets. The L-EF diet did not enhance the mitochondrial β-oxidation of fatty acids (Cpt1α and Ppar-α expressions) compared to the L-Et or L-Fr diet. Together, our data provide evidence for the coaction of ethanol and fructose with a high-fat-diet on dyslipidemia and insulin resistance-accompanied liver damage.

Figure 1. Nutrient profiles of the rat’ feeding categories.

Stacked columns show the percentage of energy contribution of the following macronutrients: protein, fat, and carbohydrates (CARB), as well as ethanol, in relevant diet category. The standard chow (Co) diet contained 58% CARB-derived calories (mainly starch). The Lieber-DeCarli (LDC) diet contained 49% calories CARB (mainly maltodextrin). In contrast, the L-Et was composed of 16% CARB-derived calories including ethanol (30%). The L-Fr diet included 45% CARB-derived calories (30% fructose). The L-EF diet contained 37% CARB (30% fructose), and 30% ethanol-derived energy. ^†indicates that each of L-Fr and L-EF diets implicates 30% J fructose, whereas in the other groups, the energy generated from fructose is negligible.

Figure 2. Animals’ phenotypes.

Growth curves of rats fed the LDC, L-Et, L-Fr or L-EF diet (A); curves of food intake (B); bars show liver weights (C); and relative liver weights (D) after the rats had been fed with different diets ad lib for 28 d. The mean values are shown with SEMs represented by vertical bars for 6 rats per group. ^abcd designate that the mean values were statistically dissimilar differences from the corresponding group vs. Co, LDC, L-Et, or L-Fr groups, respectively. Co: chow, LDC: liquid Lieber-DeCarli diet, L-Et: LDC +30% J ethanol, L-Fr: LDC +30% J fructose, L-EF: LDC+combination of 30% J ethanol and 30% J fructose.

Figure 3. Representative photomicrographs of Nile red-stained liver sections.

The stained TG are appeared as glittered, spherical, discrete droplets distributed throughout the cytoplasm (×100). The insets (×200) show hepatocytes with DAPI stained nuclei (blue) and fat droplets (green) in the experimental groups. A distinct border between zone I and the unaffected zone III was created by hepatocytes with tiny-droplet (microvesicular) steatosis in the L-EF group. PT: portal triad, Co: control, LDC: Lieber-DeCarli diet, L-Et: LDC +30% J ethanol, L-Fr: LDC +30% J fructose, L-EF: LDC+isoenergetic (30% J) ethanol and fructose.

Figure 4. Liver morphology of H&E-stained sections from each rat group after 4 wk of experimental treatment.

The LDC-fed animals developed microvesicular steatosis, and there was little or no evidence of inflammation. Both macro- and micro-steatosis were induced by the L-Et diet. In the L-Fr and L-EF regimens, zone I hepatocytes were markedly expanded predominantly by macrovesicular steatosis. Unlike zone I, towards the CV, the fat-loaded hepatocytes have central nuclei (blue arrows). CV: central vein. Original magnification×100.

Figure 5. Assessment of intrahepatic TG (A) and cholesterol (B) contents.

^a, ^aa, ^aaaindicates significant difference of the corresponding group vs. the Co (P<0.05, P<0.01, P<0.001; respectively), ^bP<0.05, ^bbP<0.01 vs. the LDC group, while ^dddenotes P<0.01 vs. the L-Fr group. The animals (n = 6) were fed for 28 d.

Figure 6. Bar graphs display plasma lipid profile.

Rats were given ad lib chow, LDC, L-Et, L-Fr or L-EF diet (n = 4). B. total cholesterol. ^aP<0.01, ^aaP<0.05 vs. respective the Co. ^bP<0.01 vs. LDC, while ***P<0.001 compared to all groups. The data are shown as mean and error bars denote SEM.

Figure 7. The bar charts show plasma glucose (A), serum leptin (B), C-peptide (C), and insulin (D) concentrations in rats after feeding with assigned diets.

The blood was collected from the rats in a fed-state at the end of wk 4. ^a, ^aadesignates the level of significant differences between the corresponding group and the Co (P<0.05, P<0.01; respectively), ^bP<0.05, ^bbP<0.01 vs. the LDC, ^cP<0.05 vs. the L-Et.

Figure 8. Quantitative analysis of gene transcription in rats fed with different Diets.

(I) Bar plots showing the relative levels of (A–F) Lep-r, Hl, Cd36, Acc2, Cpt1a, and Pparα specific mRNA transcripts in hepatic samples from 4 rats/group. PCRs were performed in duplicate. Controls were set as 1 on the y-axis. (G) The diagram shows the fold change in the Irs-1 and Irs-2 gene expressions on the logarithmic y-axis vs. feeding categories. Actb (β-actin) and Ubc were used as an internal control. Lep-r: leptin receptor, Hl: hepatic lipase, Cd36: fatty acid transporter, Acc2: acetyl-CoA carboxylase 2, Cpt1a: carnitine palmitoyltransferase Iα, Irs: insulin receptor substrate. ^aP<0.05, ^aaP<0.01, ^aaaP<0.001 vs. Co, while ^b P<0.05 ^bbP<0.01 vs. LDC, ^cP<0.05 vs. L-Et, ^dP<0.05 vs. L-Fr, ***P<0.001 compared to the remaining groups.

Figure 9. Scheme illustrating the interaction between fructose and ethanol ingested simultaneously to elicit metabolic imbalance.

The portal vein delivers the secreted insulin in addition to approximately 80% of the ingested ethanol and most of the fructose and other nutrients to the liver. Fructose and ethanol provide the metabolites that serve as the substrates for de novo lipogenesis. Fructose catabolism requires ATP to form F-1-P, glyceraldehyde, and pyruvate. The subsequent process of reducing these metabolites yields NAD⁺, a necessary coenzyme required in alcohol oxidation by ADH. An uncontrolled NADH/NAD⁺ ratio could affect mitochondrial functions. Both nutrients also induce JNK-1 activation, phosphorylating hepatic IRS-1/2 and rendering it inactive while contributing to hepatic insulin resistance. The last effect promotes hyperinsulinemia and influences the substrate’s deposition into fat, promoting hepatic steatosis, gluconeogenesis, and hyperglycemia. The liver is the major site of insulin clearance and has a first-pass clearance above 75%. Tna-α (a pro-apoptotic mediator) is expressed in such steatosic livers. ADH: alcohol dehydrogenase, IRS-1/2: insulin receptor substrate-1/2, JNK-1: c-jun N-terminal kinase, F-1-P: fructose-1-phosphate.