Transgenic Mice Overexpressing Leptin Accumulate Adipose Mass at an Older, But Not Younger, Age*

Sensitivity to leptin is associated with a normal regulation of the adipose mass, whereas decreased leptin sensitivity results in elevated adipose tissue stores. To address whether the effects of chronic hyperleptinemia are sustained with age, we generated transgenic mice that overexpress leptin under the control of the fat specific aP2 promoter/enhancer. At 6–9 weeks of age, transgenic mice overexpressed 5-fold more human leptin than endogenous mouse levels and had consistently low body weights, with reduced brown and white fat depots characterized by adipocytes either devoid of or containing minute lipid droplets. However, at 33–37 weeks, despite continuous secretion of human leptin, the transgenic mice showed a rebound effect characterized by an increase in body weight, accumulation of adipose mass, and lipid-filled adipocytes. Thus, this mouse model exhibits a two-stage phenotype, with respect to fat accumulation. In addition, plasma glucose, triglycerides, and cholesterol levels were markedly depressed in young, but not older, transgenic mice. A detrimental consequence of early hyperleptinemia was a failure of the transgenic mice to acclimatize to the cold, as a result of depleted fat stores within their brown adipocytes. Cold exposure was tolerated after a 2-week high-fat diet or at an older age when fat depots had naturally accumulated. Treatment of the older transgenic mice with large doses of leptin stimulated weight loss, demonstrating that the leptin pathway still responds to pharmacological levels of leptin. Overall, these studies show that moderate hyperleptinemia in normal mice results in a sensitivity of the adipose mass to leptin at a younger (but not older) age. The mechanisms that lead to the accumulation of fat at an older age remain largely unknown, and this hyperleptinemic mouse model will allow the uncovering of at least some of these mechanisms. (Endocrinology 142: 348–358, 2001) L A HORMONE secreted from adipose tissue (1), is the subject of intense investigations. When injected into leptin-deficient obese (ob/ob) mice, it corrects all their metabolic and physiological defects (2–6). Leptin enters the brain via a saturable transport system (7) and binds to its receptor (8) in the arcuate region of the hypothalamus, where it activates a specific signal transducer and activator of transcription (STAT-3) (9) as part of the Janus kinase signaling pathway. Animal models deficient in the synthesis of a longform of the leptin receptor, such as db/db mice and fa/fa rats (10), have a severe leptin resistance that results in an obese phenotype similar to ob/ob mice. Leptin has been implicated in various biological pathways and plays distinct roles in energy expenditure and fasting (11). Mutations in the human leptin gene are rare; however, a morbidly obese individual who was found to be homozygous for a frameshift mutation in the leptin gene (12) was successfully treated with recombinant human leptin (13). The phenotypes that result from the absence of leptin or its signaling-competent receptor prove that this pathway is not redundant and is therefore critical to the organism. Whereas common obesity in humans does not result from mutations in the leptin gene or its receptor, most obese individuals have considerably elevated circulating leptin levels (termed hyperleptinemia) as a result of increased fat mass (14). It has been hypothesized that such individuals are in a state of leptin resistance, because they fail to respond to their endogenous supraphysiological levels of leptin. Presumably, increasing the brain permeability to leptin in obese subjects would stimulate the leptin pathway via the sympathetic nervous system (15) and result in adipose tissue lipolysis. In this study, we investigated the chronic lifelong effects of hyperleptinemia in the presence of a functional and intact leptin pathway, using a transgenic animal model that expresses, in a tissue-specific fashion, a moderately elevated human leptin level. Materials and Methods Generation of transgenic mice The complementary DNA (cDNA) for human leptin spanning the initiation to termination codons was amplified by one round of RT-PCR from human adipose tissue messenger RNA (mRNA) with primers containing BamHI sites at their 39 ends. The amplified product was cleaved with BamHI and subcloned into the prokaryotic expression vector pQE30 (QIAGEN, Chatsworth, CA) to yield the plasmid hxp1.2. Human leptin cDNA was amplified from hxp1.2 with primers containing SmaI sites, and the amplified product was inserted into the SmaI site of the expression vector pBA [obtained from Dr. Richard Weiner, University of California, San Francisco (UCSF)]. The leptin cDNA was thus placed downstream of a rabbit b-globin intron and upstream of the human GH (hGH) polyadenylation (polyA) signal to generate pBA-hxp1.2. The b-globin intron, leptin cDNA, and hGH polyA sequences were then recovered as a single SacI fragment and inserted into the SacI site of pBluescript SK1. Finally, the construct was recovered from Bluescript as a SpeI fragment and ligated to the SpeI site downstream of the aP2 promoter/enhancer Bluescript-based plasmid obtained from Dr. Bruce Spiegelman (Harvard Medical School, Boston, MA). The integrity of the leptin cDNA sequence in the final construct was verified by DNA sequencing. The final construct containing the aP2 promoter/enhancer, rabbit b-globin, leptin cDNA, and hGH polyA signal (Fig. 1A) was recovered as a KpnI/NotI fragment and microinjected into C57BL6J/DBA2J embryos according to standard protocols. The presence of the transgene in founder and F1 mice was detected by SpeI digestion of tail genomic DNA followed by Southern blotting and hybridization with a P-labeled human Received July 17, 2000. Address all correspondence and requests for reprints to: F. Chehab, Department of Laboratory Medicine, 505 Parnassus Avenue, University of California, San Francisco, California 94143. E-mail: chehabf@labmed2. ucsf.edu. * This work was funded by NIH Grant HD-35142. † Supported by a postdoctoral fellowship from NIH Training Grant T32-DK-07636. 0013-7227/01/$03.00/0 Vol. 142, No. 1 Endocrinology Printed in U.S.A. Copyright © 2001 by The Endocrine Society