Limited therapeutic effect of N-acetylcysteine on hepatic insulin resistance in an experimental model of alcohol-induced steatohepatitis

Abstract

Background: Alcohol-related steatohepatitis is associated with increased oxidative stress, DNA damage, lipotoxicity, and insulin resistance in liver. As inflammation and oxidative stress can promote insulin resistance, effective treatment with antioxidants, for example, N-acetylcysteine (NAC), may restore ethanol-impaired insulin signaling in the liver.

Methods: Adult male Sprague-Dawley rats were fed for 130 days with liquid diets containing 0 or 37% ethanol by caloric content, and simultaneously treated with vehicle or NAC. Chow-fed controls were studied in parallel. Liver tissues were used for histopathology, cytokine activation, and insulin/IGF-1 signaling assays.

Results: We observed significant positive trends of increasing severity of steatohepatitis (p = 0.016) with accumulation of neutral lipid (p = 0.0002) and triglycerides (p = 0.0004) from chow to control, to the ethanol diet, irrespective of NAC treatment. In ethanol-fed rats, NAC reduced inflammation, converted the steatosis from a predominantly microvesicular to a mainly macrovesicular histological pattern, reduced pro-inflammatory cytokine gene expression, ceramide load, and acid sphingomyelinase activity, and increased expression of IGF-1 receptor and IGF-2 in liver. However, NAC did not abrogate ethanol-mediated impairments in signaling through insulin/IGF-1 receptors, IRS-1, Akt, GSK-3β, or p70S6K, nor did it significantly reduce pro-ceramide or GM3 ganglioside gene expression in liver.

Conclusions: Antioxidant treatments reduce the severity of chronic alcohol-related steatohepatitis, possibly because of the decreased expression of inflammatory mediators and ceramide accumulation, but they do not restore insulin/IGF-1 signaling in liver, most likely due to persistent elevation of GM3 synthase expression. Effective treatment of alcohol-related steatohepatitis most likely requires dual targeting of oxidative stress and insulin/IGF resistance.

Figure 1

Effects of chronic ethanol feeding and NAC treatment on steatohepatitis. Adult male Sprague-Dawley rats were fed for 130 days with chow (A, F, K), control TEN (B, G, L), control TEN supplemented with NAC (C, H, M), ethanol TEN (37% by caloric content; D, I, N), or ethanol TEN supplemented with NAC (E, J, O). Paraffin-embedded histological sections (5 µm thick) of liver were stained with H&E and photographed at 40× (A–E), 160× (F–J), and 400× (K–O) magnifications. Note reduced inflammation and shift from microvesicular steatosis to macrovesicular steatosis associated with NAC treatments, but persistence of hepatic chord architecture disarray in the control liquid + ethanol livers despite NAC treatment.

Figure 2

Lipid and ceramide accumulations in liver following chronic ethanol and NAC treatment. (A) Severity of hepatic steatosis observed in H&E stained histological sections was graded blind on a scale from 1 to 4. (B–E) Lipid extracted from fresh frozen liver tissue was analyzed for (B) neutral lipid content using the Nile Red assay, (C) triglycerides, (D) cholesterol, or (E) ceramide immunoreactivity (see Material and Methods), and values were normalized to tissue sample weight or protein content. (F) CYP2E1 expression was measured by qRT-PCR analysis. Box plots depict medians (horizontal bars), 95% confidence interval limits, and range (whiskers). Inter-group comparisons were made using one-way ANOVA with the Tukey multiple comparison and linear trend post hoc tests. Significant P-values are indicated within the panels. Significant post-hoc linear trend analysis results are shown in the upper left quadrant of each panel.

Figure 3

Role of sphingomyelinase activity as a mediator of increased hepatic ceramide levels following chronic ethanol feeding, and therapeutic effects of NAC. (A) Acid (ASMase) and (B) neutral (NSMase) sphingomyelinase activities were measured using a standard fluorometric assay and results were normalized to protein concentration. Inter-group comparisons were made using one-way ANOVA with the Tukey multiple comparison tests. Significant P-values are indicated within the panels.

Figure 4

Effects of chronic ethanol and NAC treatment on pro-inflammatory cytokine and CYP2E1 gene expression in liver. RNA extracted from liver was reverse transcribed and the cDNAs were used to measure to (A) IL-1β, (B) IL-2, (C) IL-6, (D) TNF-α, and (E) CYP2E1 by qPCR using gene-specific primers (Supplementary Table 1). Inter-group comparisons were made using one-way ANOVA with the Tukey multiple comparison tests. Significant P-values are indicated within the panels.

Figure 5

Effects of chronic ethanol and NAC treatment on insulin/IGF signaling network genes. RNA extracted from liver was reverse transcribed to measure (A) IGF-1, (B) insulin receptor, (C) IGF-1 receptor, (D) IGF-2 receptor, (E) IGF-2, (F) IRS-1, (G) IRS-2, and (H) IRS-4 mRNA expression by PCR. Inter-group comparisons were made using one-way ANOVA with the Tukey multiple comparison tests. Significant P-values are indicated within the panels.

Figure 6

Effects of chronic ethanol and NAC treatment on upstream mediators of insulin/IGF signaling. Liver protein homogenates were used to measure immunoreactivity corresponding to (A) insulin receptor (R), (B) IGF-1R, (C) IRS-1, (D) ^{pYpY1162/1163}-IR, (E) ^{pYpY1135/1136}-IGF-1R, (F) ^pS312-IRS-1 with a bead-based Multiplex ELISA platform (see Methods and Methods). (G–I) In addition, the phosphorylated/total protein ratios for were calculated to assess relative levels of phosphorylation of each protein. Data were analyzed statistically using one-way ANOVA with the Tukey multiple comparison tests. Significant P-values are indicated within the panels.

Figure 7

Effects of chronic ethanol, and NAC treatment on the downstream mediators of insulin/IGF signaling. Liver protein homogenates were used in bead-based Multiplex ELISAs to measure (A) Akt, (B) GSK-3β, (C) p70S6K, (D) PRAS40, (E) ^pS473-Akt, (F) ^pS9-GSK3β, (G) ^pTpS421/424-p70S6K and (H) ^pT246-PRAS40 immunoreactivities. (I–L) In addition, the phosphorylated/total protein ratios for were calculated to assess relative levels of phosphorylation of each protein. Data were analyzed statistically using one-way ANOVA with the Tukey multiple comparison tests. Significant P-values are indicated within the panels.