Effects and Potential Mechanisms of Pioglitazone on Lipid Metabolism in Obese Diabetic KKAy Mice

Abstract

This study aimed to analyze the effects and potential mechanisms of pioglitazone on triglyceride and cholesterol metabolism in obese diabetic KKAy mice. Pioglitazone was orally administered to KKAy mice over 30 days. Compared to C57BL/6J mice, KKAy mice developed obvious insulin resistance, hepatic steatosis, and hyperlipidemia. Pioglitazone treatment resulted in deteriorated microvesicular steatosis and elevated hepatic triglyceride levels, though plasma triglyceride and free fatty acid levels were reduced by the treatment, compared to nontreated KKAy mice. Plasma alanine aminotransferase activities were also significantly increased. Additionally, pioglitazone increased plasma concentrations of total cholesterol, HDL-cholesterol, and LDL-cholesterol but decreased hepatic cholesterol. Gene expression profiling revealed that pioglitazone stimulated hepatic peroxisome proliferator-activated receptor gamma hyperactivity, and induced the upregulation of adipocyte-specific and lipogenesis-related genes but downregulated of genes involved in triglyceride lipolysis and fatty acid β -oxidation. Pioglitazone also regulated the genes expression of hepatic cholesterol uptake and excretion, such as low density lipoprotein receptor (LDL-R) and scavenger receptor type-BI (SR-BI). These results suggested that pioglitazone could induce excessive hepatic triglyceride accumulation, thus aggravating liver steatosis and lesions in KKAy mice. Furthermore, pioglitazone may suppress the clearance of serum cholesterol from the liver predominantly through inhibition of LDL-R and SR-BI expression, thus increasing the plasma cholesterol.

Figure 1

Pioglitazone ameliorated systemic insulin resistance in diabetic KKAy mice. Fasting plasma glucose (a), insulin (b), and adiponectin (c) were measured at the end of the treatment. HOMA-IR (d) was calculated by (FPG (mg/dL) × FINS (ng/mL))/22.5. OGTT (e) and ITT (f) were performed in fasted animals on days 21 and 25 of treatment, respectively, as described in Section 2. Nor, C57BL/6J mice; Con, vehicle-treated KKAy mice; Piog, pioglitazone-treated KKAy mice. Data are presented as mean ± S.E.M. (n = 10 per group). *P < 0.05, **P < 0.01 versus Con.

Figure 2

Effects of pioglitazone on biochemical variables involved in fatty acid metabolism and tissue weight in KKAy mice. The levels of plasma TG (a), plasma FFA (b), plasma levels of AST (c), plasma levels of ALT (d), liver TG (e), and FFA (f) were measured by commercial kits. The final body weight (g), liver weight (h) and WAT weight (i) were recorded at the end of the treatment. Nor, C57BL/6J mice; Con, vehicle-treated KKAy mice; Piog, pioglitazone-treated KKAy mice. Data are presented as mean ± S.E.M. (n = 10 per group). *P < 0.05, **P < 0.01 versus Con.

Figure 3

Pioglitazone-induced effects on liver histology of KKAy mice. Representative liver photos and photomicrographs (200×) of H&E-stained liver sections from C57BL/6J mice (Nor), vehicle-treated KKAy mice (Con), and pioglitazone-treated KKAy mice (Piog).

Figure 4

Expression of adipocyte-specific genes in the liver and WAT of vehicle- and pioglitazone-treated KKAy mice. Gene expression in liver (a) and WAT (b), which was examined by using the real-time PCR. Results were expressed as fold change, after correction for β-actin levels, relative to the control mice. Nor, C57BL/6J mice; Con, vehicle-treated KKAy mice; Piog, pioglitazone-treated KKAy mice. Data are presented as mean ± S.E.M. (n = 6–8 per group). *P < 0.05, **P < 0.01 versus Con. PPARγ, peroxisome proliferator activated receptor gamma; FABP4/ap2, adipocyte fatty acid binding protein 4; LPL, lipoprotein lipase; FAT/CD36, fatty acid translocase/CD36 antigen; FSP27, fat specific protein 27; AdipoQ, adiponectin.

Figure 5

Effects of pioglitazone on cholesterol metabolism and associated hepatic protein expression in KKAy mice. The plasma TC (a), plasma HDL-C (a), plasma LDL-C (c), the levels of liver TC (d), the average food intake (e), gallbladder bile TC (f), and fecal TC (g) were measured as mentioned in Section 2. (h) Representative western blot for ABCA1, ABCG5, ABCG8, LDLR, SR-BI, and β-actin is shown. The lower left bar graph represents statistical data from six individual mice per group. The bands were determined by densitometric analysis and expressed as fold change, after correction for β-actin levels, relative to the control mice. Nor, C57BL/6J mice; Con, vehicle-treated KKAy mice; Piog, pioglitazone-treated KKAy mice. Data are presented as mean ± S.E.M. (n = 10 for biochemical analysis and n = 6 for protein analysis). *P < 0.05, **P < 0.01 versus Con.

Figure 6

Schematic diagram for the potential mechanisms of pioglitazone exacerbation of hepatic steatosis and increased plasma cholesterol in KKAy mice. Pioglitazone induced hepatic steatosis may be mediated by (1) increased uptake of FFAs from plasma to liver through promoting FFA transporter (LPL, ap2); (2) enhanced de novo lipogenesis (DNL) in the liver (FAS, ACC, SCD-1); (3) decreased TGs lipolysis and FFAs β-oxidation (HSL, CPT-1a); (4) impaired secretion of lipoprotein (VLDL) into plasma (apoB, MTTP); (5) increased dietary fat absorption. Furthermore, pioglitazone may suppress the clearance of serum cholesterol from the liver predominantly through inhibiting LDL-R and SR-BI expression, thus increasing the plasma levels of cholesterol.