Pioglitazone Ameliorates Insulin Resistance and Diabetes by Both Adiponectin-dependent and -independent Pathways

Hiroyasu Ogata ,Naoto Kubota,Kohjiro Ueki,Ryozo Nagai,Tetsuya Kubota,Masao Moroi,Hitomi Satoh ,Yasuo Terauchi,Kaoru Sugi,Shinsuke Itoh,Toshimasa Yamauchi,Wataru Yano,Kazuyuki Tobe,Mikiko Ishikawa ,Hiroki Kumagai,Kumpei Tokuyama,Iseki Takamoto,Takashi Kadowaki,Tomoka Mineyama,Tetsuo Noda

doi:10.1074/jbc.m505649200

Abstract

Thiazolidinediones have been shown to up-regulate adiponectin expression in white adipose tissue and plasma adiponectin levels, and these up-regulations have been proposed to be a major mechanism of the thiazolidinedione-induced amelioration of insulin resistance linked to obesity. To test this hypothesis, we generated adiponectin knock-out (adipo-/-) ob/ob mice with a C57B/6 background. After 14 days of 10 mg/kg pioglitazone, the insulin resistance and diabetes of ob/ob mice were significantly improved in association with significant up-regulation of serum adiponectin levels. Amelioration of insulin resistance in ob/ob mice was attributed to decreased glucose production and increased AMP-activated protein kinase in the liver but not to increased glucose uptake in skeletal muscle. In contrast, insulin resistance and diabetes were not improved in adipo-/-ob/ob mice. After 14 days of 30 mg/kg pioglitazone, insulin resistance and diabetes of ob/ob mice were again significantly ameliorated, which was attributed not only to decreased glucose production in the liver but also to increased glucose uptake in skeletal muscle. Interestingly, adipo-/-ob/ob mice also displayed significant amelioration of insulin resistance and diabetes, which was attributed to increased glucose uptake in skeletal muscle but not to decreased glucose production in the liver. The serum-free fatty acid and triglyceride levels as well as adipocyte sizes in ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were significantly reduced to a similar degree after 30 mg/kg pioglitazone. Moreover, the expressions of TNFalpha and resistin in adipose tissues of ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were decreased after 30 mg/kg pioglitazone. Thus, pioglitazone-induced amelioration of insulin resistance and diabetes may occur adiponectin dependently in the liver and adiponectin independently in skeletal muscle.

Highlights

Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan, a grant-in-aid for the Development of Innovative Technology from the Ministry of Education, Culture, Sports, Science and Technology
We found that the insulin resistance and diabetes of ob/ob mice was significantly improved in association with significant up-regulation of serum adiponectin levels after 14 days of 10 mg/kg pioglitazone treatment
Values are means Ϯ S.E. of data obtained from the analysis of ob/ob mice and adipoϪ/Ϫob/ob mice. *, p Ͻ 0.05

Summary

Animals and Genotyping

Mice were housed on a 12-h light-dark cycle and fed standard chow CE-2 (CLEA Japan Inc., Tokyo, Japan) with the following composition: 25.6% (w/w) protein, 3.8% fiber, 6.9% ash, 50.5% carbohydrates, 4% fat, and 9.2% water. To rule out the potential impact of the expression cassettes for the selection of targeted ES cells in the targeted allele on the expression of genes surrounding the adiponectin locus, selection cassettes were deleted by the Cre-Pac method as described previously [36], with some modification. We backcrossed the original adipoϪ/Ϫ mice (C57Bl/6 and 129/sv background) [24] with C57Bl/6 mice more than seven times. Ob/ob and adipoϪ/Ϫob/ob mice were prepared by adipoϩ/Ϫob/ϩ mouse intercrosses. All experiments in this study were conducted on male littermates.

Pioglitazone Treatment Study

Western Blot Analysis

Serum Adiponectin and Lipid Measurements

In Vivo Glucose Homeostasis

Measurement of Adipocyte Size

RNA Preparation and Taqman PCR

RESULTS

We previously demonstrated that TZDs increased the number of small

DISCUSSION