Stanford BIOC 230 - Physiological response to long-term peripheral and central leptin infusion in lean and obese mice

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Proc. Natl. Acad. Sci. USAVol. 94, pp. 8878–8883, August 1997PhysiologyPhysiological response to long-term peripheral and central leptininfusion in lean and obese miceJEFFREY L. HALAAS*, CAROL BOOZER†,JOHN BLAIR-WEST‡,NASEEM FIDAHUSEIN*, DEREK A. DENTON§,AND JEFFREY M. FRIEDMAN*¶i*Laboratory of Molecular Genetics and¶Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY, 10021;†Departmentof Medicine, College of Physicians and Surgeons, Columbia University, and Obesity Research Center, St. Luke’s–Roosevelt Hospital Center, New York, NY10025; and‡Department of Physiology, University of Melbourne, and§Howard Florey Institute of Experimental Physiology and Medicine, Parkville 3052,Melbourne, AustraliaContributed by Derek A. Denton, June 12, 1997ABSTRACT Recent data have identified leptin as anafferent signal in a negative-feedback loop regulating the massof the adipose tissue. High leptin levels are observed in obesehumans and rodents, suggesting that, in some cases, obesity isthe result of leptin insensitivity. This hypothesis was tested bycomparing the response to peripherally and centrally admin-istered leptin among lean and three obese strains of mice:diet-induced obese AKRyJ, New Zealand Obese (NZO), andAy. Subcutaneous leptin infusion to lean mice resulted in adose-dependent loss of body weight at physiologic plasmalevels. Chronic infusions of leptin intracerebroventricularly(i.c.v.) at doses of 3 ngyhr or greater resulted in completedepletion of visible adipose tissue, which was maintainedthroughout 30 days of continuous i.c.v. infusion. Direct mea-surement of energy balance indicated that leptin treatmentdid not increase total energy expenditure but prevented thedecrease that follows reduced food intake. Diet-induced obesemice lost weight in response to peripheral leptin but were lesssensitive than lean mice. NZO mice were unresponsive toperipheral leptin but were responsive to i.c.v. leptin. Aymicedid not respond to subcutaneous leptin and were 1y100 assensitive to i.c.v. leptin. The decreased response to leptin indiet-induced obese, NZO, and Aymice suggests that obesity inthese strains is the result of leptin resistance. In NZO mice,leptin resistance may be the result of decreased transport ofleptin into the cerebrospinal fluid, whereas in Aymice, leptinresistance probably results from defects downstream of theleptin receptor in the hypothalamus.In mammals, the maintenance of energy stores in the form oftriglycerides is of adaptive value, particularly in times ofnutritional scarcity. Numerous studies of humans and othermammals have indicated that the size of the adipose tissuemass is tightly regulated (1–3). The identification of leptin andits receptors supports this finding and indicates that leptin is anafferent signal in a negative-feedback loop regulating bodyweight (4–6). The level of plasma leptin is positively correlatedwith adipose tissue mass, and injections of recombinant leptinreduce food intake and body fat content in normal animals(7–10). Leptin injections also attenuate some of the hormonalchanges observed during starvation (11). While leptin treat-ment decreases weight in normal mice, some investigators havesuggested that this is a pharmacological effect and that leptin’sprinciple function is to signal starvation (12, 13). Recent datahave also suggested that the hypothalamus is an importanttarget of leptin action and that leptin’s metabolic effects arequalitatively different from those of food restriction (6, 14–18).Plasma leptin levels are significantly higher in obese animalsand humans (7, 19, 20). These findings have led to theconclusion that obese individuals are relatively insensitive toendogenous leptin, and as a consequence become obese (7,19). To directly test this hypothesis, the potency of subcuta-neous and intracerebroventricular (i.c.v.) infusion of leptin wascompared among lean and diet-induced obese AKRyJ (DIO),New Zealand Obese (NZO), and Aymice. The i.c.v. infusionwas performed using a microsurgical technique for long-terminfusion into the third ventricle of mice (21). AKRyJ mice arelean on a chow diet but have a heritable predisposition towarddeveloping obesity when fed a high-fat diet (22). NZO miceinherit a polygenic obesity, and Aymice carry a single domi-nant mutation that results in obesity (23–25). These strains alldevelop an obese syndrome associated with high blood levelsof leptin. The experiments reported here indicate that DIO,NZO, and Aymice are relatively insensitive to leptin. However,in each case, the sensitivity differed, providing possible insightsinto the mechanisms of leptin resistance and obesity in theseobese strains.METHODSAnimal Maintenance. Animals were purchased from TheJackson Laboratory (C57BLy6J, C57BLy6J Ay,AKRyJ) orCharles River Breeding Laboratories (NZO) and maintainedin individual cages or housed in groups of four (AKRyJ). Micewere maintained on a 12-hr light:dark schedule, fed, weighedand, if necessary, injected between 1000 and 1200. All animalswere cared for according to approved institutional guidelines.Subcutaneous Pumps. Pumps were filled aseptically with0.2-mm-filtered PBS (pH 7.4), artificial cerebrospinal fluid(aCSF) (1 mM MgCl2y1 mM CaCl2y145 mM NaCly3mMKCl), or leptin in PBS (pH 7.4) and soaked in 0.9% NaCl at37°C for 4 hr. Animals were anesthetized with methoxyfluraneand the pump was implanted subcutaneously dorsally.Intracerebroventricular (i.c.v.) Cannula. Animals wereanesthetized with ketamine and xylazine and placed on astereotaxic apparatus (Kopf Instruments model 900 smallanimal stereotaxic). The calvaria was exposed, and two 0.5-mm-diameter holes were drilled posteriorly bilaterally. Self-threading pins (TMS, Whaledent) were screwed into each holeand a 1-mm-diameter hole was drilled over bregma. Thecannula, a 30-gauge needle angled at 9°, was implanted into thethird ventricle with the following coordinates: midline, 20.3The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked ‘‘advertisement’’ inaccordance with 18 U.S.C. §1734 solely to indicate this fact.© 1997 by The National Academy of Sciences 0027-8424y97y948878-6$2.00y0PNAS is available online at http:yywww.pnas.org.Abbreviations: i.c.v., intracerebroventricular(ly); CSF, cerebrospinalfluid; aCSF, artificial CSF; DIO, diet-induced obese AKRyJ; NZO,New Zealand Obese.iTo whom reprint


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Stanford BIOC 230 - Physiological response to long-term peripheral and central leptin infusion in lean and obese mice

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