Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake
© Makimura et al; licensee BioMed Central Ltd. 2002
Received: 20 August 2002
Accepted: 7 November 2002
Published: 7 November 2002
Several lines of evidence strongly suggest that agouti-related peptide (AGRP) plays a key role in the regulation of metabolic function but ablation of the AGRP gene has no apparent effect on metabolic function. Since specific pharmacological antagonists of AGRP do not presently exist, we assessed if reduction of hypothalamic AGRP mRNA by RNA interference (RNAI) would influence metabolic function, an outcome suggesting that pharmacological antagonists might constitute useful reagents to treat obesity.
The RNAI protocol specifically reduced hypothalamic expression of AGRP mRNA by 50% and resulted in reduction of AGRP peptide immunoreactivity. Physiologically, the reduction in AGRP levels was associated with increased metabolic rate and reduced body weight without changes in food intake.
AGRP can function to increase body weight and reduce metabolic rate without influencing food intake. The present study demonstrates that RNAI protocols can be used to assess physiological function of neuronal genes in vivo.
Agouti-related peptide (AGRP) gene expression is elevated by fasting and in obesity due to leptin insufficiency [1, 2], while reversal of obese phenotypes by adrenalectomy reverses the elevation of AGRP mRNA . Injections of synthetic analogs of AGRP, and of AGRP itself, also stimulate food intake and body weight [4, 5]. Together with the observation that transgenic over-expression of the AGRP gene leads to hyperphagia and obesity , these data suggest that antagonism of AGRP may reduce food intake and body weight, thus potentially serving as a therapy for obesity.
RNAI produces sequence-specific reduction of gene expression in various mammalian in vitro systems  and most recently, RNAI was shown to suppress the expression and activity of a luciferase transgene in adult mice , suggesting that RNAI may become a valuable molecular and possibly clinical tool for use in vivo. A major advantage of RNAI over a similar protocol, using antisense oligonucleotides, is that the use of antisense oligonucleotides to assess chronic effects requires constant or repetitive infusion, is expensive and generally impractical for more than a few days. In contrast, RNAI can be used to study long-term phenotypes (including body weight and memory functions) due to the development of a DNA plasmid-based system to deliver short double-stranded RNA sequences in vivo. While RNAI has been extensively used to assess physiological functions of specific genes in C. elegans and Drosophila [9, 10] and a single recent paper has reported that RNAI can reduce gene expression in vivo (in liver) , the applicability of RNAI to assess physiological or neuronal function in mammals has not yet been demonstrated.
Here we show, using both small interfering RNA (siRNA) particles and the plasmid based RNAI delivery system, pSUPER-RNAI, that decreasing endogenous hypothalamic AGRP levels in the central nervous system leads to significant physiological responses including an increase in metabolic rate and a decrease in body weight.
Results and Discussion
Effects of small interfering RNA particles against AGRP
The decrease in AGRP mRNA and increase in metabolic rate was associated with a trend to decrease body weight, but this effect was not significant over the 48 hours after injection (body weight loss of -1.72 ± 0.66 g for the GFPi control mice compared to a loss of -2.85 ± 0.52 g for the AGRPi mice). Surprisingly, decrease in AGRP mRNA did not significantly influence food intake compared to controls (mean intake of 5.0 ± 0.7 g/day for the GFPi control mice compared to 5.3 ± 0.6 g/day for the AGRPi mice). Since both the AGRPi and the control GFPi mice were still recovering from the effects of the surgery and anesthesia at 48 hours, it is not surprising that the effects on body weight were not statistical at this time. Nevertheless the lack of an effect on food intake was unexpected since treatment with synthetic AGRP peptides has profound effects on food intake [4, 5].
Effects of the plasmid based RNAI protocol against AGRP
In summary, the present report represents the first demonstration that RNA interference can be used to reduce expression of an endogenous gene in adult mammals. This protocol has allowed us to assess the physiological function of an endogenous neuronal gene. This study also directly indicates that hypothalamic AGRP regulates metabolic rate. The observation that reduction of AGRP mRNA would produce more profound effects on metabolic rate than on food intake was surprising since infusion of AGRP peptide produces profound effects on food intake [4, 5]. However, since neither AGRP mRNA nor peptide was completely ablated, it seems likely that effects on food intake would become more apparent with a more dramatic manipulation. Although ablation of the AGRP gene was reported to have no effect on metabolic phenotype , our results suggest that complete ablation of the gene may have produced metabolic compensation. Thus reducing (but not ablating) AGRP expression in adult mice may have identified a metabolic role for AGRP not apparent from complete ablation of the gene, suggesting that agents antagonizing the effect of AGRP may be useful to treat obesity without producing unacceptable loss of appetite. Consistent with previous findings in mammalian systems [6–8] and unlike other systems such as C. elegans and Drosophila [9, 10], RNAI reduced but did not eliminate expression of AGRP. However, improvements in delivery systems, especially in conjunction with viral-based gene transfer protocols , could result in greater and even longer-term reduction of target gene expression suggesting that RNAI will prove to be a useful tool to assess physiological functions of mammalian genes in vivo, especially those expressed in the brain, as RNAI has been so useful in functional mapping of genes in non-mammalian systems.
Animal and RNAI protocols
The appropriate Institutional Animal Review Board had approved all studies. To study the effects of RNAI, C57Bl/6J mice retired breeders were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were individually housed with free access to food and water under 12:12 h light-dark cycle (lights on at 07:00 h). Stereotaxic surgery was performed under anesthesia (2.5% 2-2-2-tribromoethanol, 0.015–0.017 ml/g body weight, i.p.). The RNAI constructs were injected bilaterally using a 1 ul Hamilton syringe placed on a stereotaxic frame into the following coordinates from bregma: anterior-posterior -0.12; lateral ± 0.02; vertical -0.6. Double-stranded siRNA particles to reduce expression of AGRP were designed based on Elbashir et al.  (Fig. 1a). The siRNA oligonucleotides were designed from the beginning of exon 1 of both AGRP and GFP genes and ordered gel-purified and annealed from Oligos Etc. (Wilsonville, OR). SiRNA particles were re-suspended in Rnase-free water to a concentration of 1 mM and 0.5 ul was injected over 2 minutes by stereotaxic surgery bilaterally. The pSUPER-RNAI plasmid constructs were re-suspended in lipofectin (Invitrogen Corp. Carlsbad, CA) to a concentration of 1 ug/ul, and 1 ul of the mixture was injected bilaterally over five minutes using the same coordinates as for the siRNA particles. Body weight and food intake was monitored daily.
Metabolic rate was monitored continuously by indirect-calorimetry for three days following surgery. Air from each cage was sampled every five minutes, and oxygen and carbon dioxide concentrations from each sample were measured independently. From the change in oxygen, VO2 or oxygen consumption can be calculated after normalizing to body weight. Heat production can then be calculated using the following formula: Heat (cal/hour) = ((4.33+0.67 × (VCO2/VO2)) × VO2 × (Body weight) × 60). Therefore, the parameter VO2 is normalized to body weight, whereas heat production is not normalized to body weight.
For Northern blot analysis, all mice were sacrificed toward the end of the light period (between 17:00 and 19:00 h) by decapitation after a brief exposure to carbon dioxide. Brains were quickly removed and the hypothalamus was dissected out, frozen on dry ice and stored at -70°C until use. For immunocytochemistry, mice were perfused with cold PBS followed by 4% paraformaldehyde. Brains were removed, post-fixed in paraformaldehyde then preserved in 30% sucrose solution for 48 hours.
Northern blot analysis
Total RNA was extracted from tissue using TRIzol (GIBCO BRL, Gaitherburg, MD). Concentration of the samples were measured by UV spectrophotometer and normalized to 1 ug/ul. Uniform concentration and integrity of the samples were verified by agarose gel electrophoresis prior to Northern blot analysis. Six micrograms of total RNA from hypothalamus was subjected to Northern blot analysis, as described previously, to measure AGRP mRNA [15, 16, 2, 3]. Briefly, RNA was denatured by incubating with glyoxal and dimethyl sulfoxide for 1 hour at 50°C and separated on a 1.5% agarose 10 mM NaPO4 (pH = 7.0) gel for 1 hour at 100 V. The RNA was then transferred to an Immobilon S (Millipore) membrane by capillary elution overnight in 20 × standard saline citrate (SSC) buffer. Membranes were briefly washed in 6 × SSC, baked at 80°C and cross-linked by UV light. The membranes were prehybridized in Ultrahyb buffer (Ambion) at 68°C for 2 hours and hydribized overnight with 3.5 × 106 DPM/ml of probe at 42°C. The blots were probed with single-stranded internally labeled DNA probe as described previously . The membranes were washed twice in 1 × SSC/0.1% SDS solution for 15 minutes at room temperature, followed by 0.1 × SSC/0.1% SDS solution for 15 minutes at room temperature twice and then for 3 hours at 42°C. The membranes were then exposed to phosphoimager screen overnight. The total integrated densities of hybridization signals were determined by phosphoimager (STORM 860, Molecular Dynamics, Sunnyvale, CA). To monitor RNA loading, membranes were re-probed and hybridized with a 32P-labeled probe encoding 18S ribosomal RNA.
Cryopreserved brains were cut to a thickness of 30 um in a cryostat at -20°C and stored in PBS-B buffer (10 mM PO4 in 0.9% NaCl) containing 0.1% sodium azide at 4°C. Immunocytochemistry was performed with a rabbit polyclonal primary antibody to AGRP peptide diluted 1 to 10,000 (Phoenix Pharmaceuticals, Belmont, CA) followed by Vectastain ABC kit (Vector Laboratories, Burlingame, CA). The immunoreactivity was visualized with the Vector VIP kit (Vector Laboratories, Burlingame, CA). Sections were mounted on microscopic slides, air-dried and cover slipped with VectaMount (Vector Laboratories, Burlingame, CA).
Statistical analysis for the Northern blot was performed by student's t-test, using the JMP statistical package implemented on the Macintosh operating system. A p-value of less than 0.05 was considered significant. For the analysis of the metabolic cage data, a two-way ANOVA (time × RNAI construct) was performed using the JMP statistical package. For analysis of food intake and body weight, a two-way ANOVA (time × RNAI construct) followed, where indicated, by student's t-test (p < 0.05 was considered significant) was performed to compare each independent time point.
The authors would like to thank I-Wei Shu for his expertise in immunocytochemistry and Chineta L. Pullin for her help in cloning.
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