Figure 7. KLF4 is required for the increase in AgRP expression following GsD stimulation in AgRP neurons.

(a) Differential effects on hypothalamic KLF4, AgRP and NPY mRNA expression following hM3Dq or GsD stimulation in AgRP neurons. GsD-AgRP and hM3Dq-AgRP mice were treated with CNO (1 mg kg⁻¹ i.p.), and hypothalamic RNA was prepared 2 h later. Gene expression levels were determined via real-time quantitative reverse transcription–PCR (qRT–PCR). Expression data were normalized relative to results obtained with saline-injected GsD-AgRP mice (control mice). (b) Activation of GsD expressed in AgRP neurons of AgRP-ires-Cre mice selectively stimulates KLF4 protein expression in GsD-expressing neurons. The AAV-hSyn-DIO-GsD-eGFP virus was unilaterally injected into the ARC of AgRP-ires-Cre (right side). The GsD receptor was detected by using an anti-GFP antibody that recognizes the eGFP tag fused to the C terminus of GsD. Hypothalamic slices were processed for immunostaining studies 1 h after CNO administration (1 mg kg⁻¹ i.p.). Scale bar, 200 μm. (c) Effects of kenpaullone (Ken), an inhibitor of KLF4 expression, on CNO-dependent changes in gene expression in GsD-AgRP mice. GsD-AgRP mice were first injected (i.c.v.) with either saline (−) or Ken (2 μg). Thirty minutes later, all mice were injected with CNO (1 mg kg⁻¹ i.p.). Hypothalamic RNA was isolated and processed for qRT–PCR studies 2 h after CNO treatment. Experiments were carried out with 11- to 17-week-old male mice. Data are given as means±s.e.m. (n=3–5 mice per group).*P<0.05, **P<0.01, ***P<0.001, as compared with saline-treated GsD-AgRP mice (a) or GsD-AgRP mice pretreated with i.c.v. saline (c), respectively. (d) Immunohistochemical detection of KLF4 expression in AgRP neurons of AgRP-ires-Cre mice injected with the AAV-hSyn-DIO-KLF4 virus. KLF4 was visualized by using an anti-HA antibody that recognizes the HA tag that was fused to the C terminus of KLF4. Scale bar, 200 μm. (e) Selective expression of KLF4 in AgRP neurons leads to enhanced food intake. To obtain KLF4-AgRP and control mice, AgRP-ires-Cre mice were injected bilaterally with the AAV-hSyn-DIO-KLF4 virus or the AAV-hSyn-DIO-mCherry virus, respectively. Experiments were carried out with 10-week-old male mice. Data are given as means±s.e.m. (n=6 mice per group).***P<0.001, as compared with the control group (a,c,e, Student's t-test).