Pioglitazone improves lipid and insulin levels in overweight rats on a high cholesterol and fructose diet by decreasing hepatic inflammation

Abstract

Background and purpose: Nutrient overload leads to obesity and insulin resistance. Pioglitazone, a selective peroxisome proliferator-activated receptor (PPAR)gamma agonist, is currently used to manage insulin resistance, but the specific molecular mechanisms activated by PPARgamma are not yet fully understood. Recent studies suggest the involvement of suppressor of cytokine signalling (SOCS)-3 in the pathogenesis of insulin resistance. This study aimed to investigate the hepatic signalling pathway activated by PPARgamma activation in a non-genetic insulin-resistant animal model.

Experimental approach: Male Wistar rats were maintained on a high-cholesterol fructose (HCF) diet for 15 weeks. Pioglitazone (3 mg x kg(-1)) was administered orally for the last 4 weeks of this diet. At the end of the treatment, serum was collected for biochemical analysis. Levels of PPARgamma, SOCS-3, pro-inflammatory markers, insulin receptor substrate-2 and Akt/glycogen synthase kinase-3beta phosphorylation were assessed in rat liver.

Key results: Rats fed the HCF diet exhibited hyperlipidemia, hyperinsulinemia, impaired glucose tolerance and insulin resistance. Pioglitazone administration evoked a significant improvement in lipid metabolism and insulin responsiveness. This was accompanied by reduced hepatic expression of SOCS-3, interleukin-6, tumour necrosis factor-alpha and markers of neutrophil infiltration. Diet-induced PPARgamma expression was unaffected by the pioglitazone treatment.

Conclusion and implications: Chronic pioglitazone administration reduced hepatic inflammatory responses in rats fed a HCF diet. These effects were associated with changes in hepatic expression of SOCS-3, which may be a crucial link between the reduced local inflammation and the improved insulin signalling.

Figure 1

Effects of two dietary regimens either normal (Control) or a high-cholesterol diet with 10% fructose solution (HCF) on oral glucose tolerance in rats treated with pioglitazone (Pio; 3 mg·kg⁻¹, p.o.) (Control+Pio; HCF+Pio). Values are mean ± SD of 6–9 animals per group. P < 0.05 versus Control.

Figure 2

Alterations in hepatic expression of PPARγ induced by diet and pioglitazone administration. PPARγ protein expression was measured by Western blot analysis in liver homogenates of rats fed with a standard (Control) or HCF diet in the absence or presence of pioglitazone (Pio) treatment (Control+Pio; HCF+Pio). Densitometric analysis of the bands is expressed as relative optical density (O.D.), corrected for the corresponding β-actin and normalized using the related Control band. Data are means ± SD of three separate experiments. P < 0.01 versus Control.

Figure 3

Effects of pioglitazone on insulin signal transduction in the liver of rats fed a HCF diet. IRS-2 (A), total Akt protein expression and Ser473 phosphorylation (B), and total GSK-3β protein expression and Ser9 phosphorylation (C) were analysed by Western blot on the liver obtained from Wistar rats fed with a standard (Control) or HCF diet for 15 weeks and treated with pioglitazone (Pio; 3 mg·kg⁻¹, p.o.) during the last 4 weeks (Control+Pio; HCF+Pio). Densitometric analysis of the bands is expressed as relative optical density (O.D.), corrected for the corresponding β-actin contents and normalized using the related Control band. The data are means ± SD of three separate experiments. P < 0.01 versus Control.

Figure 5

Pioglitazone prevents diet-induced expression of inflammatory markers and neutrophil infiltration in the rat liver. COX-2 (A), iNOS (B) and ICAM-1 (D) protein expression was measured by Western blot analysis in liver homogenates of rats fed with a standard (Control) or a HCF diet in the absence or presence of pioglitazone (Pio) treatment (3 mg·kg⁻¹, p.o.) (Control+Pio; HCF+Pio). Densitometric analysis of the bands is expressed as relative optical density (O.D.), corrected for the corresponding β-actin contents and normalized using the related Control band. Myeloperoxidase (MPO) activity (C) was measured by spectrophotometric analysis from rat liver homogenates. Data are means ± SD of three separate experiments for Western Blot and five animals per group for MPO. P < 0.01 versus Control.

Figure 4

Effects of pioglitazone on SOCS-3 and cytokine expression in the rat liver. mRNA expression levels of SOCS-3 (A), IL-6 (B) and TNF-α (C) were analysed by RT-PCR in the livers of rats fed with a standard (Control) or a HCF diet in the absence or presence of pioglitazone (Pio) treatment (3 mg·kg⁻¹, p.o.) (Control+Pio; HCF+Pio). Densitometric analysis of the bands is expressed as relative optical density (O.D.), corrected for the corresponding 18S contents and normalized using the related Control band. Data are means ± SD of three separate experiments. P < 0.01 versus Control.

Figure 6

Pioglitazone induces the expression of established target genes of PPARγ. mRNA expression levels of LGK, FAS and PCG-1α were analysed by RT-PCR in the livers of rats fed with a HCF diet in the absence or presence of pioglitazone (Pio) treatment (3 mg·kg⁻¹, p.o.) (HCF+Pio). 18S content was used as housekeeping gene for normalization of RNA quantization. Each gel photograph is from a single experiment and is representative of three separate experiments.