Transgenic Neuronal Expression of Proopiomelanocortin Attenuates
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Transgenic Neuronal Expression of Proopiomelanocortin Attenuates Hyperphagic Response to Fasting and Reverses Metabolic Impairments in Leptin-Deficient Obese Mice Tooru M. Mizuno,1,2,3 Kevin A. Kelley,1,4 Giulio M. Pasinetti,1,5 James L. Roberts,1 and Charles V. Mobbs1,2,3 Hypothalamic proopiomelanocortin (POMC) gene ex- pression is reduced in many forms of obesity and diabe- tes, particularly in those attributable to deficiencies in any forms of obesity and diabetes are associ- leptin or its receptor. To assess the functional signifi- ated with impairments in the production of cance of POMC in mediating metabolic phenotypes as- (1–5), processing of (6,7), or sensitivity to sociated with leptin deficiency, leptin-deficient mice M(8,9) products of the proopiomelanocortin bearing a transgene expressing the POMC gene under (Pomc) gene. In particular, ablation of leptin or its recep- control of the neuron-specific enolase promoter were tor causes reduced hypothalamic expression of POMC as produced. The POMC transgene attenuated fasting- well as obesity and impairments in glucose metabolism induced hyperphagia in wild-type mice. Furthermore, (1–3). Fasting also leads to reduced leptin and reduced the POMC transgene partially reversed obesity, hy- expression of hypothalamic POMC (3). Similarly, hypotha- perphagia, and hypothermia and effectively normalized lamic POMC expression is reduced in other conditions hyperglycemia, glucosuria, glucose intolerance, and in- involving impairments in glucose metabolism caused by sulin resistance in leptin-deficient mice. Effects of the POMC transgene on glucose homeostasis were indepen- hypothalamic lesions (10), mutations in the tubby and dent of the partial correction of hyperphagia and obe- nhlh2 genes (11,12), aging (13), or insulin deficiency (14). sity. Furthermore, the POMC transgene normalized the The suggestion that reduced POMC may actually be a profile of hepatic and adipose gene expression associ- cause of the metabolic impairments comes from the ated with gluconeogenesis, glucose output, and insulin observation that mutations in the Pomc gene lead to sensitivity. These results indicate that central POMC is metabolic impairments in humans (4) and mice (5). a key modulator of glucose homeostasis and that ago- It is plausible that the POMC-derived peptide ␣-melano- nists of POMC products may provide effective therapy in cyte-stimulating hormone (MSH) mediates many of the treating impairments in glucose homeostasis when hy- effects of hypothalamic POMC on metabolic function, as pothalamic POMC expression is reduced, as occurs with central injection of ␣-MSH or an ␣-MSH agonist decreases leptin deficiency, hypothalamic damage, and aging. food intake and increases energy expenditure (8,15,16), Diabetes 52:2675–2683, 2003 whereas central injection of a melanocortin receptor an- tagonist increases food intake and decreases energy ex- penditure (17–19). Because the blockade of ␣-MSH signaling blocks the acute effects of leptin on feeding behavior (20), ␣-MSH may mediate some of leptin’s meta- From the 1Fishberg Center for Neurobiology, Mount Sinai School of Medicine, bolic effects and therefore reduced hypothalamic POMC New York, New York; 2Neurobiology of Aging Laboratories, Mount Sinai School of Medicine, New York, New York; the 3Department of Geriatrics, may mediate effects of leptin deficiency on metabolic and Mount Sinai School of Medicine, New York, New York; the 4Department of neuroendocrine impairments. Accumulating genetic data Molecular, Cellular, and Developmental Biology, Mount Sinai School of also support a role for the melanocortin system in the Medicine, New York, New York; and the 5Department of Psychiatry, Mount Sinai School of Medicine, New York, New York. regulation of glucose metabolism. Agouti mice develop Current affiliation for J.L.R. is Department of Pharmacology, University of obesity, hyperphagia and diabetes with hyperinsulinemia, Texas Health Science Center at San Antonio, San Antonio, Texas. Address correspondence and reprint requests to Charles V. Mobbs, PhD, and glucose intolerance because of the ectopic expression Neurobiology of Aging Laboratories, Box 1639, Mount Sinai School of Medi- of the agouti peptide, which acts as an antagonist to cine, 1 Gustave L. Levy Pl., New York, NY 10029. E-mail: charles.mobbs@ ␣-MSH (21–24). Mice with disruption of the ␣-MSH recep- mssm.edu. Received for publication 16 April 2003 and accepted in revised form 1 tor melanocortin 4 receptor (MC4-R) exhibit a phenotype August 2003. similar to that observed in agouti mice, including obesity, AGRP, agouti-related peptide; FAT, fatty acid transporter; FFA, free fatty acid; GTG, gold-thio-glucose; MC4-R, melanocortin 4 receptor; MSH, melano- hyperphagia, and hyperinsulinemia (9). However, whether cyte-stimulating hormone; NPY, neuropeptide Y; Nse, neuron specific enolase; perturbed glucose homeostasis is primarily caused by PGC-1, peroxisome proliferatorϪactivated receptor-␥ coactivator 1; POMC, decreased melanocortin tone or secondary to obesity is proopiomelanocortin; PTP-1B, protein tyrosine phosphatase 1B; SCD-1, stearoyl-CoA desaturase 1; SOCS-3, suppressor of cytokine signaling 3. unknown at present. That impairments in the POMC © 2003 by the American Diabetes Association. system might directly cause insulin resistance is suggested DIABETES, VOL. 52, NOVEMBER 2003 2675 CENTRAL POMC AND GLUCOSE HOMEOSTASIS by the observation that mice with impaired sensitivity to hyperphagia was assessed in Pomcϩ/ϩ and Pomcϩ/tg mice at age 7 months. ␣-MSH are highly insulin resistant, possibly independent Mice were fasted for 24 h by removing food immediately before lights out (at ␣ 1900), and 24 h later, mice were allowed free access to food by reintroducing of adiposity (25,26). These data suggest that -MSH ago- preweighed food. Food intake was measured after access to the food for 1 h nists might be effective in improving diabetic phenotypes after lights out. As a control, food intake was also measured for 1 h after lights such as insulin resistance and hyperglycemia. On the other out in mice of both genotypes fed ad libitum. hand, infusion of ␣-MSH agonists directly into the brain Pair-feeding. Lepob/ob mice were pair-fed to the level of food intake of age-matched Pomcϩ/tg Lepob/ob mice for 2 weeks (between ages 20 and 22 causes marked impairments in glucose tolerance while weeks). Half the amount of food consumed during the previous 24 h by reducing insulin secretion (26), suggesting that ␣-MSH Pomcϩ/tg Lepob/ob was presented to Pomcϩ/ϩ Lepob/ob mice at 0900 and the agonists might worsen diabetic symptoms, at least acutely. other half at 1600 to prevent effects of prolonged fasting on blood glucose Although considerable evidence suggests that reduced levels. Blood glucose was measured at 1800. At the end of the pair-feeding hypothalamic POMC might contribute to metabolic impair- period, mice were fasted overnight and a glucose tolerance test was per- formed as described below. ments in many forms of obesity and diabetes, these Measurement of metabolic rate. The metabolic rate was determined using metabolic impairments involve altered expression of many an indirect calorimetry system (29). In the morning, mice were placed into genes. Therefore, a key question is whether elevation of metabolic cages (Accuscan, Columbus, OH) for6htodetermine metabolic rate and activity. Oxygen consumption (VO ) and heat production was POMC gene expression can reverse the metabolic impair- 2 calculated per 5-min period. Data during the last2hofmeasurement were ments in conditions otherwise associated with reduced used for the analysis to avoid responses attributable to a novel environment. hypothalamic POMC gene expression. Thus the present ob/ob ϩ/ϩ Because Lep mice are substantially heavier than Lep mice, VO2 may study assessed whether chronic enhancement of central reflect differences in the mass of nonmetabolically active tissue and differ- POMC expression attenuates metabolic impairments asso- ences in the metabolic rate of metabolically active tissue. To address this ciated with leptin deficiency. The results demonstrated issue, total oxygen consumption during the 2-h experimental period was also calculated. that elevation of POMC gene expression in neurons mark- Glucose tolerance and insulin tolerance tests. Glucose tolerance tests edly attenuates fasting-induced hyperphagia in wild-type were performed at age 12 weeks. Insulin tolerance tests were performed mice, obesity and hyperphagia in leptin-deficient Lepob/ob between ages 21 and 25 weeks. For the glucose tolerance test, mice were mice, and impairments in glucose metabolism in Lepob/ob fasted overnight (15 h) by removing their food immediately before lights out (1900) and injecting them intraperitoneally with glucose (2 mg/g body wt) at mice independent of effects on food intake or body weight. 1000. A drop of blood was taken from the tail vein immediately before glucose injection and 15, 30, 60, 120, and 180 min after injection. Plasma insulin concentrations during the glucose tolerance test were also measured. Mice RESEARCH DESIGN AND METHODS were fasted overnight and injected intraperitoneally with glucose (2 mg/g Animals. Mice were weaned 3–4 weeks after birth and fed regular rodent diet body wt). Blood samples were collected from the tail vein every 15 min for 1 h. (4.02 kcal/g, 11.9% kcal from fat; #5,053, Purina Mills, St. Louis, MO) and water For insulin tolerance tests, human insulin (Novolin; Novo Nordisk, Princeton, thereafter. Mice were individually