Evidence for the Existence of Distinct Central Appetite, Energy Expenditure, and Ghrelin Stimulation Pathways as Revealed by Hypothalamic Site-Specific Leptin Gene Therapy

0013-7227/02/$15.00/0 Printed in U.S.A. Endocrinology 143(11):4409 – 4421 Copyright © 2002 by The Endocrine Society doi: 10.1210/en.2002-220505 Evidence for the Existence of Distinct Central Appetite, Energy Expenditure, and Ghrelin Stimulation Pathways as Revealed by Hypothalamic Site-Specific Leptin Gene Therapy M. BAGNASCO, M. G. DUBE, P. S. KALRA, AND S. P. KALRA Departments of Neuroscience (M.B., S.P.K.) and Physiology and Functional Genomics (M.G.D., P.S.K.), McKnight Brain Institute, University of Florida, Gainesville, Florida 32610-0244 To identify the specific hypothalamic sites in which leptin acts to decrease energy intake and/or increase energy expenditure, recombinant adeno-associated virus vector-encoding leptin was microinjected bilaterally into one of four hypothalamic sites in female rats. Leptin transgene expression in the ventromedial nucleus and paraventricular nucleus induced comparable decreases in daily food intake (FI; 18 –20%) and body weight (BW; 26 –29%), accompanied by drastic reductions in serum leptin (81–97%), insulin (92–93%), free fatty acids (35–36%), and normoglycemia. Leptin transgene expression in the arcuate nucleus (ARC) decreased BW gain (21%) and FI (11%) to a lesser range, but the metabolic hormones were suppressed to the same extent. Leptin transgene expression in the medial preoptic area (MPOA) decreased BW and metabolic L EPTIN PRODUCED BY adipocytes and hypothalamus (1– 4) controls the daily management of body weight (BW) homeostasis by restraining food intake (FI) and enhancing energy expenditure (5–7). A loss of leptin control on these two central mechanisms invariably results in uncontrolled energy intake leading to obesity and attendant metabolic disorders such as hyperleptinemia, hyperinsulinemia, and type 2 diabetes (5– 8). Numerous studies now suggest that despite the presence of elevated circulating leptin levels, the progressive age-related and environmentally based increase in adiposity is due to leptin insufficiency in the hypothalamus rendered by defective transport of peripheral leptin across the blood brain barrier and/or suboptimal production of leptin locally in the hypothalamus (9 –14). Gene delivery in vivo to the central nervous system has been facilitated by the development of a nonimmunogenic and nonpathogenic recombinant adeno-associated virus (rAAV) vector (15, 16). The rAAV has advantages over other viral vector systems because of availability of stable, hightiter vector for long-term expression of target genes in nondividing cells (15–17). Consequently, leptin gene therapy Abbreviations: AgrP, Agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; BW, body weight; FFA, free fatty acid; FI, food intake; GFP, green fluorescence protein; icv, intracerebroventricular; ISH, in situ hybridization; MPOA, medial preoptic area; NPY, neuropeptide Y; PF, pair fed; POMC, proopiomelanocortin; PVN, paraventricular nucleus; rAAV, recombinant adeno-associated virus; rAAVlep, rAAV-vector encoding the leptin transgene; ROD, relative OD; UCP1, uncoupling protein-1; VMN, ventromedial nucleus. hormones without decreasing FI. Finally, leptin transgene expression in all four sites augmented serum ghrelin and thermogenic energy expenditure, as shown by uncoupling protein-1 mRNA expression in brown adipose tissue. Proopiomelanocortin gene expression in the ARC was up-regulated by leptin expression in all four sites, but neuropeptide Y gene expression in the ARC was suppressed by leptin transgene expression in the ARC but not in the MPOA. Thus, whereas leptin expression in the paraventricular nucleus, ventromedial nucleus, or ARC suppresses adiposity and insulin by decreasing energy intake and increasing energy expenditure, in the MPOA it suppresses these variables by increasing energy expenditure alone. (Endocrinology 143: 4409 – 4421, 2002) offers a novel way to reinstate the hypothalamic leptin insufficiency responsible for the age-related and environmentally based abnormal weight gain and adiposity. We recently developed a rAAV-vector encoding the leptin transgene (rAAV-lep) (18). A single intracerebroventricular (icv) injection of this vector inhibited weight gain and adiposity for long periods in rats of both sexes maintained either on regular rat chow or high-fat diet (10, 11, 19, 20). Interestingly, in association with suppressed weights, these rats displayed drastic reductions in serum leptin, insulin, and free fatty acids (FFAs) along with normoglycemia. In addition, icv rAAV-lep augmented thermogenic energy expenditure alone or along with decreased FI (10, 11, 19 –22). The physiologically active long form of the leptin receptor is expressed in various hypothalamic sites, and leptin administration to rodents activates c-Fos protein in groups of neurons in multiple hypothalamic sites (5, 23–27), suggesting that receptive elements in these sites play a role in regulating energy balance. Experimental results showed that microinjection of leptin into several hypothalamic sites decreased food intake (28, 29). These sites include the arcuate nucleus (ARC)-paraventricular nucleus (PVN) axis in which leptin receptors are expressed in neurons expressing the orexigenic peptides, neuropeptide Y (NPY), and agouti- related peptide (AgrP) and in proopiomelanocortin (POMC) neurons producing the anorexigenic peptide, ␣-MSH (5, 6, 30, 31). We have now extended our icv leptin gene therapy studies to ascertain whether intracranial delivery of rAAV-lep in distinct hypothalamic sites would transduce leptin-trans- 4409 4410 Endocrinology, November 2002, 143(11):4409 – 4421 Bagnasco et al. • Central Leptin Gene Therapy gene. Because the evidence outlined above suggests multiple sites of leptin action in weight control, another goal of these studies was to determine whether leptin transgene expression is one or more than one hypothalamic site that will reproduce the effects of icv rAAV-lep administration on agerelated weight gain and metabolic hormones. In view of the fact that leptin administration inhibits FI and increases energy expenditure, it was of interest to identify the site(s), if any, in which leptin expression would selectively modulate either energy intake or energy expenditure and whether peptidergic pathways in the ARC-PVN axis are affected by the site-specific leptin transgene expression. treated, rAAV-UF5, and rAAV-lep, six to eight rats per group). Rats were anesthetized with ketamine ⫹ Xylazine, a blood sample (1 ml) was withdrawn from the jugular vein, and plasma was stored frozen for analysis. Rats were then microinjected bilaterally either rAAV-lep or rAAV-UF5 (1013 particles/ml) in the ARC (0.3 ␮l/injection, 3 ⫻ 109 particle/nuclei, at 2.7 mm behind the bregma, 0.2 mm lateral to the sagittal sinus, and 9.8 mm below the dura) or in the medial preoptic area (MPOA) (0.3 ␮l/injection, 3 ⫻ 109 particle/nuclei, at 0.3 mm behind the bregma, 0.6 mm lateral to the sagittal sinus, and 7.8 mm b (...truncated)