Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity

Abstract

Obesity is a major public health problem and is often associated with type 2 diabetes mellitus, cardiovascular disease, and metabolic syndrome. Leptin is the crucial adipostatic hormone that controls food intake and body weight through the activation of specific leptin receptors (OB-R) in the hypothalamic arcuate nucleus (ARC). However, in most obese patients, high circulating levels of leptin fail to bring about weight loss. The prevention of this "leptin resistance" is a major goal for obesity research. We report here a successful prevention of diet-induced obesity (DIO) by silencing a negative regulator of OB-R function, the OB-R gene-related protein (OB-RGRP), whose transcript is genetically linked to the OB-R transcript. We provide in vitro evidence that OB-RGRP controls OB-R function by negatively regulating its cell surface expression. In the DIO mouse model, obesity was prevented by silencing OB-RGRP through stereotactic injection of a lentiviral vector encoding a shRNA directed against OB-RGRP in the ARC. This work demonstrates that OB-RGRP is a potential target for obesity treatment. Indeed, regulators of the receptor could be more appropriate targets than the receptor itself. This finding could serve as the basis for an approach to identifying potential new therapeutic targets for a variety of diseases, including obesity.

Fig. 1.

OB-RGRP interacts with OB-R and regulates its cell surface expression. (A) HeLa cells were transduced with Ad:OB-Ra and increasing doses of Ad:OB-RGRP. Cell surface proteins were biotinylated and isolated with streptavidin-Sepharose beads. Surface (biotinylated) over total OB-R and transferrin receptor were plotted as a function of Ad:OB-RGRP dose. (B) Effect of OB-RGRP-specific antisense (AS1, AS2) or control (AS3, AS4) oligonucleotides on endogenous OB-RGRP mRNA levels in HeLa cells monitored by semiquantitative RT-PCR. GAPDH was used as an internal control. (C) Effect of AS2 (OB-RGRP-specific) or AS4 (control) on the cell surface expression of the indicated receptors. NT, nontransfected HeLa cells expressing lower levels of endogenous OB-Ra. (D) Coimmunoprecipitation of OB-Ra and OB-RGRP. Cells expressing the indicated proteins were lysed (input) and subjected to immunoprecipitation (IP) and Western blotting (WB) with the indicated antibodies. (E) Interaction of OB-Ra and OB-RGRP studied by BRET in intact cells. BRET donor saturation curves were generated by expressing constant amounts of OB-Ra-Luc and increasing quantities of the indicated YFP-tagged proteins. The BRET, total luminescence, and total fluorescence were measured. Data obtained for the BRET acceptor OB-RGRP-YFP were best-fitted with a nonlinear regression equation assuming a single binding site, those obtained for insulin receptor (IR)-YFP were best-fitted with a linear regression equation. (F) Effect of AS2 or AS4 on the activation of a STAT3 reporter gene construct by leptin in HeLa cells. (G) Quantification of STAT3 reporter gene activity in HeLa cells expressing increasing amounts of surface expressed OB-Rb. Results are means ± SE of three independent experiments performed in duplicate (*, P < 0.05).

Fig. 2.

OB-RGRP silencing in the ARC. (A) Infection of murine Ltk cells with control or OB-RGRP-specific shRNA expressing lentivirus. Endogenous OB-RGRP mRNA levels were monitored by semiquantitative RT-PCR 2 days after infection. GAPDH was used as an internal control. (B) Detection of OB-RGRP and OB-Rb mRNA levels in the ARC by real-time PCR (10 animals per condition). (C and D) Leptin-promoted STAT3 phosphorylation in the ARC from mice that have been injected with control or OB-RGRP shRNA expressing virus by stereotactic injection (D, means + SE of 12 animals per condition). *, P < 0.05.

Fig. 3.

Experimental groups and lentiviral targeting of the ARC. (A) Experimental protocol. (B) Immunohistological detection of the expression of the GFP reporter gene in the ARC.

Fig. 4.

Effect of OB-RGRP silencing in the ARC on DIO in mice. (A) Body weight evolution of mice expressing control shRNA or OB-RGRP shRNA in the ARC and fed a LFD or HFD. (B) Comparison of body weight of mice expressing OB-RGRP shRNA outside and in the ARC and fed a LFD or HFD. (C–F) Determination of fat mass (C), plasma leptin (D), insulin (E), and glucose (F) levels for the indicated experimental groups. *, P < 0.05.