Pioglitazone prevents obesity-related airway hyperreactivity and neuronal M2 receptor dysfunction

Abstract

Obesity-related asthma often presents with more severe symptoms than non-obesity-related asthma and responds poorly to current treatments. Both insulin resistance and hyperinsulinemia are common in obesity. We have shown that increased insulin mediates airway hyperreactivity in diet-induced obese rats by causing neuronal M₂ muscarinic receptor dysfunction, which normally inhibits acetylcholine release from parasympathetic nerves. Decreasing insulin with streptozotocin prevented airway hyperreactivity and M₂ receptor dysfunction. The objective of the present study was to investigate whether pioglitazone, a hypoglycemic drug, prevents airway hyperreactivity and M₂ receptor dysfunction in obese rats. Male rats fed a low- or high-fat diet were treated with pioglitazone or PBS by daily gavage. Body weight, body fat, fasting insulin, and bronchoconstriction and bradycardia in response to electrical stimulation of vagus nerves and to aerosolized methacholine were recorded. Pilocarpine, a muscarinic receptor agonist, was used to measure M₂ receptor function. Rats on a high-fat diet had potentiated airway responsiveness to vagal stimulation and dysfunctional neuronal M₂ receptors, whereas airway responsiveness to methacholine was unaffected. Pioglitazone reduced fasting insulin and prevented airway hyperresponsiveness and M₂ receptor dysfunction but did not change inflammatory cytokine mRNA expression in alveolar macrophages. High-fat diet, with and without pioglitazone, had tissue-specific effects on insulin receptor mRNA expression. In conclusion, pioglitazone prevents vagally mediated airway hyperreactivity and protects neuronal M₂ muscarinic receptor function in obese rats.

Keywords: M2 muscarinic receptors; asthma; insulin; obesity; pioglitazone.

Figure 1.

Experiment protocol, animal body weight, and food intake. A: obese-prone rats were fed a high-fat diet or low-fat diet for 5 wk. Animals on a high-fat diet were treated with either pioglitazone (3 mg/kg) or PBS (control) by daily gavage. After 5 wk, all rats were fasted overnight (16 h) and airway physiology was tested the next day. B: rats were weighed before and every week during the diet treatment. High-fat diet-fed rats gained more body weight than that in low-fat diet-fed rats, but pioglitazone did not prevent the increase of body weight gain induced by high-fat diet. C: the percentage of body fat was significantly higher in rats fed a high-fat diet than that in rats fed a low-fat diet, and pioglitazone did not prevent the increase of body fat induced by a high-fat diet. Although the food intake every week was higher in rats on a low-fat diet (D), calories consumed every week (E) was not significantly different. n = 5–16. *P < 0.05.

Figure 2.

Fasting glucose and insulin. After 5 wk of a low-fat diet or high-fat diet and treated with pioglitazone (3 mg/kg) or PBS by daily gavage, rats were fasted overnight and blood glucose (A) and plasma insulin (B) were measured the following day. n = 5–18. *P < 0.05 compared with all other groups.

Figure 3.

Airway physiology in vagotomized rats. A airway responsiveness was measured by electrically stimulating the vagus nerves with increasing frequency to cause frequency-dependent bronchoconstriction (measured as an increase in pulmonary inflation pressure) in vagotomized, anesthetized rats. B airway smooth muscle function was determined by measuring increased pulmonary inflation pressure in response to increasing concentrations (1–30 mM) of aerosolized methacholine (MCh) delivered in 20 µL saline in vagotomized, anesthetized rats. n = 4–6. *P < 0.05.

Figure 4.

Bradycardia induced by vagus nerve stimulation (A) and by inhaled methacholine (B) in vagotomized, anesthetized rats. Rats were fed a low-fat or high-fat diet and either treated with pioglitazone (3 mg/kg) or PBS by daily gavage for 5 wk. There was a nonsignificant trend toward greater bradycardia in response to vagal stimulation in high-fat diet-fed rats compare with low-fat diet-fed rats, regardless of pioglitazone treatment (A). #P = 0.0934. Inhaled MCh-induced bradycardia was not different among groups of rats (B). n = 4–6.

Figure 5.

Neuronal M₂ muscarinic receptor function was measured in vagotomized, anesthetized rats. Rats were fed a low-fat or high-fat diet and either treated with pioglitazone (3 mg/kg) or PBS by daily gavage for 5 wk. M₂ muscarinic receptor function on parasympathetic nerves was measured using pilocarpine, a muscarinic receptor agonist that is physiologically selective for M₂ over M₃ receptors. Bronchoconstriction was measured in response to electrical stimulation of both vagus nerves (5 V, 10 Hz, for 6 s at 40-s intervals). There was no difference in vagally induced bronchoconstriction among groups before administration of pilocarpine (A). Pilocarpine dose dependently inhibited vagally induced bronchoconstriction. Pilocarpine-induced inhibition was significantly reduced in rats on a high-fat diet (filled circles); it was not reduced in high-fat diet rats treated with pioglitazone (filled triangles; B). n = 4–6. #P = 0.0029; *P = 0.0161.

Figure 6.

Cytokine and insulin receptor mRNA expression in alveolar macrophages. Alveolar macrophages were isolated from bronchoalveolar lavage fluid collected from rats fed a low-fat diet (LFD) or a high-fat diet (HFD) and treated with pioglitazone (3 mg/kg) or PBS by daily gavage for 5 wk. Tumor necrosis factor-α (TNF-α; A), transforming growth factor-β (TGF-β; B), interleukin (IL)-1β (C), IL-6 (D), and insulin receptor (IR; E) mRNA were quantified by real-time RT-PCR. n = 5–8. *P < 0.05.

Figure 7.

Quantification of total insulin receptor and insulin receptor isoform mRNA expression. Adipose, brain, and lung tissues were collected from rats that were fed either a low-fat diet (LFD) or a high-fat diet (HFD) and treated with pioglitazone (3 mg/kg) or PBS by daily gavage for 5 wk. mRNA was isolated from adipose (A and D), brain (B and E), and lung (C and F) tissue. Total insulin receptor expression (A–C), insulin receptor isoform A (IR-A), and insulin receptor isoform B (IR-B) mRNA expression (D–F) were quantified using real-time RT-PCR. n = 5–8. *P < 0.05 between treatment groups; #P < 0.05 between IR-A and IR-B within the same treatment group.