Adenoviral-mediated modulation of Sim1 expression in the paraventricular nucleus affects food intake

Abstract

Haploinsufficency of Sim1, which codes for a basic helix-loop-helix-PAS (PER-ARNT-SIM) transcription factor, causes hyperphagia in mice and humans, without decrease in energy expenditure. Sim1 is expressed in several areas of the brain, including the developing and postnatal paraventricular nucleus (PVN), a region of the hypothalamus that controls food intake. We have previously found that the number of PVN cells is decreased in Sim1+/- mice, suggesting that their hyperphagia is caused by a developmental mechanism. However, the possibility that Sim1 functions in the postnatal PVN to control food intake cannot be ruled out. To explore this hypothesis, we used adenoviral vectors to modulate Sim1 expression in the postnatal PVN of wild-type mice. Unilateral stereotaxic injection into the PVN of an adenoviral vector producing a short hairpin RNA directed against Sim1 resulted in a significant increase in food intake, which peaked to 22% 6 d after the procedure, compared with the injection of a control virus. In contrast, injection of an adenovirus that expresses Sim1 induced a decrease in food intake that was maximal on the seventh day after the procedure, reaching 20%. The impact of bilateral injections of these vectors into the PVN was not greater than that of unilateral injections. Together, these results strongly suggest that Sim1 functions along a physiological pathway to control food intake.

Figure 1.

Western blot detection of SIM1 production in cultured 293A cells infected with adenoviruses. A, Production of SIM1 protein was detectable in cells infected with Ad-Sim1 but not with Ad-CTRL. B, Cells were infected with Ad-Sim1 (at a constant MOI of 5) and/or Ad-shRNA-Sim1 (at an MOI of 10 or 100). Coinfection with Ad-Sim1 and Ad-shRNA-Sim1 resulted in a 40% decrease in SIM1 production when an MOI of 10 of the latter virus was used, whereas infection with Ad-shRNA-Sim1 at an MOI of 100 decreased SIM1 production by 60%. Infection by these viruses did not affect actin production. C, Cells were infected with Ad-Sim1 (at a constant MOI of 5) and/or Ad-CTRL (at an MOI of 10 or 100). Infection with Ad-CTRL did not affect the production of SIM1 protein by the Ad-Sim1 virus.

Figure 2.

Unilateral infection of the PVN with the Ad-shRNA-Sim1 virus increases food intake. A, Daily food intake of mice infected with Ad-CTRL or Ad-shRNA-Sim1 (∗∗p < 0.005; n = 8 mice for each virus). B–D, Representative coronal sections through the PVN of a mouse infected with Ad-shRNA-Sim1. The virus was injected into the right side of the PVN, and the mouse was killed 8 d after the infection. B and C are bright- and dark-field views, respectively, of the same section, which was hybridized with a Sim1 probe, whereas D is adjacent to B and C and shows RFP production. The arrow in B–D is positioned on the tract of the needle with its pointed extremity corresponding to the deepest end of the tract. E, Quantification of Sim1 expression by in situ hybridization. The Ad-CTRL or Ad-shRNA-Sim1 virus was always injected on the right side. The sum of intensity values for each side of the PVN was compared. The values corresponding to the injected side are expressed relatively to those corresponding to the noninjected side, which are set at 100% (∗∗p < 0.005; n = 8 mice for each side). Error bars indicate SEM.

Figure 3.

Unilateral infection of the PVN with the Ad-Sim1 virus decreases food intake. A, Daily food intake of mice infected with the Ad-CRTL or the Ad-Sim1 virus (∗∗p < 0.005; n = 7 mice for each virus). B–D, Representative coronal sections through the PVN of a mouse that was infected with the Ad-Sim1. The virus was injected into the right side of the PVN, and the mouse was killed 8 d after the infection. B and C are bright- and dark-field views, respectively, of the same section, which was hybridized with a Sim1 probe, whereas D is adjacent to B and C and shows GFP production. The arrow in C and D is positioned on the tract of the needle with its pointed extremity corresponding to the deepest end of the tract. E, Quantification of Sim1 expression by in situ hybridization. The Ad-CTRL or the Ad-Sim1 virus was always injected on the right side. The sum of intensity values for each side of the PVN was compared. The values corresponding to the injected side are expressed relatively to those corresponding to the noninjected side, which are set at 100% (∗p < 0.005; n = 8 mice for each side). Error bars indicate SEM.

Figure 4.

Bilateral infection of the PVN with adenoviruses that modulate Sim1 expression levels affects food intake. Daily food intake of mice infected with the Ad-Sim1, Ad-shRNA-Sim1, or the Ad-CTRL virus (∗p < 0.05; ∗∗p < 0.005; n = 7 mice for each virus). Error bars indicate SEM.