Pioglitazone does not improve insulin signaling in mice with GH over-expression

Abstract

Type 2 diabetes and obesity are very serious health problems in both developed and developing countries. An increased level of GH is known to promote insulin resistance. Transgenic (Tg) mice over-expressing bovine GH are short-living and characterized, among other traits, by hyperinsulinemia and increased insulin resistance in comparison with normal (N) mice. Pioglitazone (PIO) is a member of the thiazolidinediones - a group of insulin-sensitizing drugs that are selective agonists of peroxisome proliferator-activated receptor gamma (PPARγ). The aim of the study was to analyze the effects of PIO on the insulin-signaling pathway in Tg and N mice. Plasma levels of insulin and glucose as well as hepatic levels of proteins involved in insulin signaling were analyzed by ELISA or western blot methods. Treatment with PIO decreased plasma level of glucose in N mice only. Similarly, PIO increased insulin sensitivity (expressed as the relative insulin sensitivity index; RISI) only in N mice. In the liver, PIO decreased the phosphorylation of insulin receptor substrate-1 (IRS1) at a serine residue (Ser(307)-pS-IRS1), which inhibits insulin action, and had a tendency to increase tyrosine phosphorylation of IRS2 (Tyr-pY-IRS2) only in N mice but did not affect either of these parameters in Tg mice. Levels of total and phosphorylated mammalian target of rapamycin were increased in Tg mice. Moreover, the level of AKT2 was decreased by PIO in N mice only. In conclusion, the lack of improvement of insulin sensitivity in insulin-resistant Tg mice during PIO treatment indicates that chronically elevated GH levels can inhibit the beneficial effects of PIO on insulin signaling.

Keywords: growth hormone; insulin signaling; pioglitazone; transgenic mice.

Fig. 1

Plasma insulin level [mg/dl] before (A) and after (B) pioglitazone (PIO) treatment, blood glucose level [mg/dl] before (C) and after (D) PIO treatment in normal (N) and transgenic mice over-expressing bovine growth hormone (Tg). Values are means ± SEM. a, b – values that do not share the same letter in the superscript are significantly different (p<0.05).

Fig. 2

The Relative Insulin Sensitivity Index (RISI) [100/√ blood glucose level x insulin level] before (A) and after (B) pioglitazone (PIO) treatment in normal (N) and transgenic mice over-expressing bovine growth hormone (Tg). Values are means ± SEM. a, b – values that do not share the same letter in the superscript are significantly different (p<0.05).

Fig. 3

Plasma adiponectin level [mg/dl] before (A) and after (B) pioglitazone (PIO) treatment in normal (N) and transgenic mice over-expressing bovine growth hormone (Tg). Values are means ± SEM. a, b, c – values that do not share the same letter in the superscript are significantly different (p<0.05).

Fig. 4

(A) Hepatic total insulin receptor (IR) protein level [units/100 μg protein] in normal (N) and transgenic mice over-expressing bovine growth hormone (Tg) without pioglitazone (PIO) treatment, and after PIO treatment in normal (N-Pio) and transgenic mice over-expressing bovine growth hormone (Tg-Pio); (B) hepatic insulin receptor substrate-2 (IRS2) phosphorylated at a tyrosine residue (IRS2-T pY) protein level [units/300 μg protein] in N mice treated with saline (N-sal) or insulin (N-ins) and Tg treated with saline (Tg-sal) or insulin (Tg-ins) without PIO treatment, and after PIO treatment in N mice treated with saline (N-P-sal) or insulin (N-P-ins) and Tg mice treated with saline (Tg-P-sal) or insulin (Tg-P-ins); (C) hepatic total insulin receptor substrate-1 (IRS1) protein level (using primary antibody from Cell Signaling Technology, Inc., Danvers, MA) in N and Tg mice without pioglitazone (PIO) treatment, and after PIO treatment in N (N-Pio) and Tg mice (Tg-Pio), (D) hepatic insulin receptor substrate-1 (IRS1) phosphorylated at a serine(307) residue (IRS1-ser-pY) protein level (using primary antibody from Cell Signaling Technology, Inc., Danvers, MA) in N and Tg mice without pioglitazone (PIO) treatment, and after PIO treatment in N (N-Pio) and Tg mice (Tg-Pio). Values are means ± SEM. a, b, c – values that do not share the same letter in the superscript are significantly different (p<0.05).

Fig. 5

(A) Hepatic total mTOR protein level (using primary antibody from Cell Signaling Technology, Inc., Danvers, MA) in normal (N) and transgenic mice over-expressing bovine growth hormone (Tg) without pioglitazone (PIO) treatment, and after PIO treatment in normal (N-Pio) and transgenic mice over-expressing bovine growth hormone (Tg-Pio); (B) hepatic mTOR phosphorylated at a tyrosine residue (mTOR-pY) protein level (using primary antibody from Cell Signaling Technology, Inc., Danvers, MA) in N and Tg mice without pioglitazone (PIO) treatment, and after PIO treatment in N (N-Pio) and Tg mice (Tg-Pio); (C) hepatic AKT2 protein level (using primary antibody from Cell Signaling Technology, Inc., Danvers, MA) in N and Tg mice without pioglitazone (PIO) treatment, and after PIO treatment in N (N-Pio) and Tg mice (Tg-Pio). Values are means ± SEM. a, b, c – values that do not share the same letter in the superscript are significantly different (p<0.05).