PPAR Agonists Reverse Ethanol-Induced Liver Pathology
Livers from control rats (liquid diets and chow) exhibited the expected well-organized lobular architecture with minimal evidence of steatosis, variation in nuclear size, or hepatocyte drop-out (). In contrast, ethanol exposed livers had microvesicular and macrovesicular steatosis with multiple foci of intralobular lympho-mononuclear inflammatory cell infiltrates, scattered areas of apoptosis and/or necrosis (), and hepatic architectural disarray with loss of regular chords and increased variability in size of hepatocyte nuclei. There was no evidence of increased fibrosis, regenerating nodule formation, or cirrhosis in ethanol-exposed livers (Gomori trichrome stain). Control rats treated with the PPAR-α agonist had no detectable histological changes in liver () relative to vehicle-treated controls. In contrast, control rats treated with the PPAR-δ () or PPAR-γ () agonist had less well-organized hepatic architecture due to sinusoidal widening and apparently increased hepatocyte crowding. In addition, PPAR-δ or PPAR-γ agonist treatments resulted in increased nuclear prominence and micro-vacuolation of hepatocyte cytoplasm. The micro-vacuolation was associated with increased PAS staining, corresponding to glycogen accumulation (data not shown). Among ethanol-fed rats, treatment with PPAR agonists had variable effects on liver histology in terms of reducing the architectural disarray, steatosis, and cell death (). Treatment with the PPAR-α, PPAR-δ, or PPAR-γ agonist reduced the disordered architecture and resulted in more of a chord-like arrangement of hepatocytes, and both micro- and macrosteatosis were reduced. Nonetheless, small foci of necrosis (, insets), inflammation (), and apoptosis (, inset) were still readily detected, although these lesions were less conspicuous than in vehicle-treated, ethanol-exposed livers. The most striking improvements in liver histology occurred in ethanol-fed rats that had been treated with the PPAR-δ () or PPAR-γ () agonist.
Figure 1 Peroxisome-proliferator activated receptor (PPAR) agonists produce subtle changes in normal liver histology: Adult male Long Evans rats were pair-fed for 8 weeks with isocaloric liquid diets containing 0% or 37% ethanol by caloric content. After 3 weeks (more ...)
Figure 2 Peroxisome-proliferator activated receptor (PPAR) agonists partially reverse ethanol-induced liver histopathology: Adult male Long Evans rats were pair-fed for 8 weeks with isocaloric liquid diets containing 0% or 37% ethanol by caloric content. After (more ...)
PPAR agonist treatments alter cell population profiles in liver
Liver mRNA levels of albumin, apical sodium-dependent bile transporter protein (ASBT), glial fibrillary acidic protein (GFAP), Kupffer cell receptor (KCR), desmin, and collagen were measured by qRT-PCR analysis. Albumin expression was used as an index of hepatocyte abundance/function. ASBT reflects bile duct epithelium. GFAP is an early marker of stellate cell activation and desmin marks transdifferentiation of hepatic stellate cells into myofibroblasts. KCR is a maker of Kupffer cells and increased expression could reflect an intra-hepatic response to injury. Collagen gene expression corresponds to fibrogenic potential or active fibrogenesis. Altogether, these assessments of gene expression, which we have termed “cell profiling”, enabled us to quantify ethanol and PPAR agonist associated shifts in liver cell type and function. In previous studies, we used this approach to characterize differential in vivo effects of chronic ethanol exposure or gene delivery on survival and proliferation of specific cell populations in relation to disease (5
Albumin expression was similar in livers of control and ethanol fed vehicle- or PPAR-α agonist-treated rats (). Treatment with the PPAR-δ or PPAR-γ agonist increased albumin expression in control livers, resulting in significantly higher levels of albumin mRNA relative to the corresponding ethanol-exposed livers (both P<0.001).
Figure 3 Peroxisome-proliferator activated receptor (PPAR) agonist treatments alter cell population profiles in liver: Control and chronic ethanol-fed rats were treated twice weekly by i.p. injection of vehicle, or a PPAR-α, PPAR-δ, or PPAR-γ (more ...)
ASBT expression was significantly higher in vehicle-treated control livers relative to all other groups (P<0.001). Among rats treated with a PPAR-agonist, ASBT expression was similar for corresponding control and ethanol-exposed groups ().
KCR expression was lowest in vehicle-treated control and ethanol-exposed livers. In the control group, treatment with a PPAR agonist did not significantly alter KCR expression. In ethanol exposed livers, KCR expression was significantly increased by treatment with the PPAR-α (P<0.05), PPAR-δ (P<0.01), or PPAR-γ (P<0.01) agonist. However, there were no significant differences in KCR expression between control and ethanol-fed rats within corresponding treatment groups ().
GFAP is an early marker of stellate cell activation. Hepatic GFAP mRNA levels were not significantly different between control and ethanol-fed rats, and the levels were not significantly altered by PPAR agonist treatments ().
Desmin is an intermediate filament expressed in stellate cells during transdifferentiation into myofibroblast-like cells. Desmin expression was similar in vehicle-treated control and ethanol-exposed livers. Among control rats, desmin expression was significantly reduced by PPAR-δ agonist treatment relative to vehicle (P<0.05). In addition, PPAR-γ agonist treatment significantly increased desmin expression in ethanol-exposed relative to corresponding control livers (P<0.01) ().
Collagen gene expression reflects fibrogenesis. Collagen mRNA levels were similar for each of the corresponding control and ethanol groups (). However, collagen expression was reduced by PPAR-δ agonist treatment in ethanol-fed rats, although the difference resulting from that response did not reach statistical significance (P=0.08).
Effects of ethanol and PPAR agonists on hepatic expression of insulin and IGF polypeptides, their receptors, and IRS molecules
QRT-PCR studies demonstrated expression of insulin, IGF-1, IGF-2 polypeptide genes, their corresponding receptors, and IRS-1, IRS-2, and IRS-4 in livers of both control and ethanol-fed rats (), indicating that the upstream genes required for insulin and IGF signaling are all expressed in adult rat livers. Among the polypeptide genes, insulin was least abundant, followed by IGF-2; IGF-1 was most abundantly expressed ().
Figure 4 Ethanol and Peroxisome-proliferator activated receptor (PPAR) agonists minimally alter expression of genes required for insulin and IGF signaling. Effects of ethanol and PPAR agonist treatments on hepatic mRNA expression of (a) insulin, (b) insulin-like (more ...)
Insulin gene expression was similar in vehicle-treated control and ethanol-exposed livers. Although PPAR-δ and PPAR-γ agonists increased insulin expression in control rats, and PPAR-α and PPAR-δ agonists reduced insulin expression in ethanol-fed rats, the inter-group differences did not reach statistical significance ().
IGF-1 mRNA levels were similar in vehicle-treated control and ethanol-exposed livers (). PPAR agonist treatments did not significantly change IGF-1 expression relative to vehicle among control or ethanol-fed rats. However, the mean hepatic levels of IGF-1 were significantly lowered by PPAR-δ (P<0.001) or PPAR-γ (P<0.01) treatment of ethanol-fed relative to corresponding control rats.
Hepatic IGF-2 mRNA levels were similar for corresponding vehicle- or PPAR-agonist treated control and ethanol-exposed rats (). However, PPAR-γ treatment significantly reduced IGF-2 expression relative to vehicle (P<0.05) among ethanol-fed rats, whereas among controls, PPAR agonist treatments did not significantly alter hepatic IGF-2 expression.
Insulin receptor expression was similar among controls treated with vehicle or a PPAR agonist, whereas among ethanol-fed rats, insulin receptor was significantly reduced following PPAR-δ agonist treatment relative to vehicle, PPAR-α, and PPAR-γ treatment (P<0.05) (). In addition, insulin receptor expression was significantly reduced in ethanol-fed, PPAR-δ agonist treated relative corresponding controls (P<0.01), whereas for the other treatment groups, there were no significant differences between control and ethanol associated levels of hepatic insulin receptor expression.
IGF-1 receptor expression was higher in vehicle-treated, ethanol-fed versus control rats (P<0.05) (). PPAR agonist treatments had no significant effect on IGF-1R expression among control rats. In contrast, PPAR-α (P<0.01) and PPAR-δ (P<0.001) agonist treatments significantly down-regulated IGF-1R expression in livers of ethanol-fed rats. In addition, IGF-1R expression was also lower in ethanol+PPAR-γ treated rats, but the difference from vehicle treatment did not reach statistical significance. Consequently, the mean hepatic IGF-1R mRNA levels were similar in control and ethanol-fed rats that received the same treatments.
IGF-2 receptor expression was highest in vehicle-treated control and ethanol-fed rats. Treatment with PPAR agonists reduced hepatic IGF-2R expression in both control and ethanol-fed rats, but the differences relative to vehicle treatment were significant only with respect to PPAR-α treatment (). There were no significant differences in the mean levels of IGF-2R between similarly treated control and ethanol-exposed livers ().
In livers of both control and ethanol-exposed rats, IRS-1 mRNA levels were highest (), followed by IRS-2 (), and then IRS-4 (). The mean levels of IRS-1 were similar for control and ethanol-fed rats (). There were no significant differences in the mean levels of IRS-1, IRS-2, or IRS-4 between control and ethanol exposed rats that had similar treatments. However, modest reductions (not statistically significant) in IRS-1, IRS-2, and IRS-4 expression occurred in ethanol-fed rats that were treated with the PPAR-δ or PPAR-γ agonist ().
Effects of ethanol and PPAR agonist treatments on insulin and IGF receptor binding
Competitive saturation binding assays were used to demonstrate the effects of ethanol and PPAR agonist treatments on insulin, IGF-1 and IGF-2 receptor binding. Binding curves ± 95% C.I.L. (data not shown), computations of Kd (dissociation constant; affinity) and BMAX (top-level binding), and inter-group statistical comparisons () were generated with Prism Graphics 5 software. In all experimental conditions, a single-site model produced the highest R2, i.e. best fit. Among controls, the BMAX and Kd for insulin receptor binding were similar following vehicle, PPAR-δ, or PPAR-γ treatment, whereas both were significantly reduced after PPAR-α treatment relative to vehicle (). Among ethanol fed rats, the BMAX and Kd for insulin receptor binding were similar, irrespective of treatment. However, the BMAX and Kd were significantly reduced relative to correspondingly treated controls, except for PPAR-α treatment, which significantly increased the BMAX for insulin receptor binding in ethanol fed rats ().
Among controls, PPAR-α and PPAR-δ agonist treatments reduced the BMAX for IGF-1R binding, the PPAR-α agonist also reduced the Kd, whereas the PPAR-γ agonist increased both the BMAX and Kd. Chronic ethanol feeding (vehicle treated) significantly reduced the BMAX (P<0.05) and Kd (P<0.01) for IGF-1R binding relative to control (+vehicle), but those adverse effects of ethanol were rescued by PPAR agonist treatments ().
For IGF-2R binding, the BMAX was similar in livers from vehicle and PPAR-α-treated controls, but significantly reduced in control rats that had been treated with the PPAR-δ or PPAR-γ agonist (both P<0.001). The Kd's were similar for all but PPAR-γ treated group, in which it was significantly reduced relative to control (P<0.01). Chronic ethanol feeding reduced the BMAX of IGF-2R binding relative to vehicle-treated control (P<0.05). Treatment with PPAR agonists restored IGF-2R binding, and resulted in similar BMAX levels in livers of ethanol-fed and corresponding controls. In contrast, the Kd's of IGF-2R binding were not significantly altered by ethanol feeding, irrespective of treatment ().
Effects of ethanol and PPAR agonist treatments on insulin/IGF responsive gene expression related to energy metabolism and tissue remodeling
AAH expression increases with insulin, IGF-1, or IGF-2 stimulation (25
), and has positive effects on hepatocellular growth and motility (25
). GAPDH is an insulin-responsive gene that has an important role in glucose metabolism. Western blot analysis detected AAH, GAPDH, and β-actin expression in all samples (). Re-probing the blots with monoclonal antibody to β-actin demonstrated approximately equal protein loading in all lanes. Digital image quantification of the Western blot signals revealed similar levels of AAH (), GAPDH (), and β-actin () expression across all control groups, irrespective of PPAR agonist treatment. Chronic ethanol feeding (+ vehicle treatment) reduced AAH (P<0.05) and GAPDH (P<0.05), but not β-Actin expression relative to control. The PPAR agonist treatments slightly increased AAH immunoreactivity; this effect abolished the statistical significance of the inter-group differences. In contrast, GAPDH expression remained similarly reduced in livers of ethanol-fed rats, although the differences from corresponding controls were not significant for the PPAR-δ and PPAR-γ treated rats. β-actin expression was similar in control and ethanol-fed rats that received similar treatments. These results were confirmed by ELISA studies (data not shown).
Figure 5 Peroxisome-proliferator activated receptor (PPAR) agonists increase insulin and insulin-like growth factor (IGF) responsive gene expression in ethanol-exposed livers. Effects of ethanol and PPAR agonist treatments on insulin/IGF responsive gene expression (more ...)