Diabetes-causing gene, kruppel-like factor 11, modulates the antinociceptive response of chronic ethanol intake

Abstract

Background: Alcohol (EtOH [ethanol]) is an antinociceptive agent, working in part, by reducing sensitivity to painful stimuli. The transcription factor Kruppel-like factor 11 (KLF11), a human diabetes-causing gene that also regulates the neurotransmitter metabolic enzymes monoamine oxidase (MAO), has recently been identified as an EtOH-inducible gene. However, its role in antinociception remains unknown. Consequently, we investigated the function of KLF11 in chronic EtOH-induced antinociception using a genetically engineered knockout mouse model.

Methods: Wild-type (Klf11(+/+) ) and KLF11 knockout (Klf11(-/-) ) mice were fed a liquid diet containing EtOH for 28 days with increasing amounts of EtOH from 0% up to a final concentration of 6.4%, representing a final diet containing 36% of calories primarily from EtOH. Control mice from both genotypes were fed liquid diet without EtOH for 28 days. The EtOH-induced antinociceptive effect was determined using the tail-flick test before and after EtOH exposure (on day 29). In addition, the enzyme activity and mRNA levels of MAO A and MAO B were measured by real-time RT-PCR and enzyme assays, respectively.

Results: EtOH produced an antinociceptive response to thermal pain in Klf11(+/+) mice, as expected. In contrast, deletion of KLF11 in the Klf11(-/-) mice abolished the EtOH-induced antinociceptive effect. The mRNA and protein levels of KLF11 were significantly increased in the brain prefrontal cortex of Klf11(+/+) mice exposed to EtOH compared with control Klf11(+/+) mice. Furthermore, MAO enzyme activities were affected differently in Klf11 wild-type versus Klf11 knockout mice exposed to chronic EtOH. Chronic EtOH intake significantly increased MAO B activity in Klf11(+/+) mice.

Conclusions: The data show KLF11 modulation of EtOH-induced antinociception. The KLF11-targeted MAO B enzyme may contribute more significantly to EtOH-induced antinociception. Thus, this study revealed a new role for the KLF11 gene in the mechanisms underlying the antinociceptive effects of chronic EtOH exposure.

Keywords: Antinociceptive Response; Chronic Ethanol Intake; Gene Knockout; Kruppel-Like Factor 11 (Transforming Growth Factor-Beta-Inducible Early Gene 2); Mice; Monoamine Oxidase.

Figure 1

Effects of chronic ethanol intake on antinociceptive response (anti-pain effect) in the wild type (Klf11^+/+) and KLF11 knockout (Klf11^−/−) mice. Mice were fed an ethanol diet or control diet for 28 days. (A) Antinociceptive effects were determined in ethanol-fed and control-fed Klf11^+/+ mice (lanes 2 vs. 1) as well as ethanol-fed and control-fed Klf1^−/− mice (lanes 4 vs. 3) by the tail-flick test. The antinociceptive effects are expressed as a percentage of the maximum possible antinociceptive effect (% MPE). (B) The base line of the antinociceptive response was also determined in four groups of Klf11^+/+ and Klf11^−/− mice before chronic ethanol exposure. Data represent the mean ± SEM of 10 mice (n = 10) in each group. * p<0.0001 and **p<0.001

Figure 2

Effects of chronic ethanol intake on the expression of KLF11 in the brain prefrontal cortex of wild type (Klf11^+/+) and knockout (Klf11^−/−) mice. Mice were fed an ethanol diet or control diet for 28 days. (A) The protein levels of KLF11 in the prefrontal cortex were examined by Western blotting. The quantitative analysis of western blot result is shown on the top. Each KLF11 protein band was evaluated by the relative intensity (relative optical density × pixel area) of its autoradiographic band and normalized to the density of β-actin. The graph of the average optical density of KLF11/actin for the individual animals (open circles or open squares) and mean values (horizontal lines) are shown with 10 mice (n = 10) for each the control group (open circles) and the ethanol-fed group (open squares). The representative western blots (bottom) show the immunolabelling of KLF11 in the prefrontal cortex of 2 untreated Klf11^+/+ controls, 2 ethanol-treated Klf11^+/+, 2 untreated Klf1^−/− controls and 2 ethanol-treated Klf11^−/− mice. The anti-β-actin antibody was used as the loading controls. (B) The mRNA levels of KLF11 in the prefrontal cortex were determined by quantitative real-time RT-PCR. Data represent the mean ± SEM of 10 mice (n = 10) in each group. * p<0.0001, **p<0.001 and ***p<0.01

Figure 3

Effects of chronic ethanol intake on the expression of monoamine oxidase (MAO) in the brain prefrontal cortex of wild type (Klf11^+/+) and knockout (Klf11^−/−) mice. Mice were fed an ethanol diet or control diet for 28 days. (A) The mRNA (a) and enzyme activity (b) of MAO A and (B) the mRNA (a) and enzyme activity (b) of MAO B in the prefrontal cortex were examined by Real-time RT-PCR and catalytic activity assays, respectively. Data represent the mean ± SEM of 10 mice (n = 10) in each group. Aa, p<0.02 (lanes 3 vs. 1 and lanes 4 vs. 2); Ab, p<0.07 (lanes 4 vs. 2); Ba, p<0.02 (lanes 3 vs. 1) and p<0.05 (lanes 4 vs. 2); Bb, p<0.02 (lanes 3 vs. 1)