Disruption of peripheral leptin signaling in mice results in hyperleptinemia without associated metabolic abnormalities.

Although central leptin signaling appears to play a major role in the regulation of food intake and energy metabolism, the physiological role of peripheral leptin signaling and its relative contribution to whole-body energy metabolism remain unclear. To address this question, we created a mouse model (Cre-Tam mice) with an intact leptin receptor in the brain but a near-complete deletion of the signaling domain of leptin receptor in liver, adipose tissue, and small intestine using a tamoxifen (Tam)-inducible Cre-LoxP system. Cre-Tam mice developed marked hyperleptinemia (approximately 4-fold; P < 0.01) associated with 2.3-fold increase (P < 0.05) in posttranscriptional production of leptin. Whereas this is consistent with the disruption of a negative feedback regulation of leptin production in adipose tissue, there were no discernable changes in energy balance, thermoregulation, and insulin sensitivity. Hypothalamic levels of phosphorylated signal transducer and activator of transcription 3, neuropeptide expression, and food intake were not changed despite hyperleptinemia. The percentage of plasma-bound leptin was markedly increased (90.1-96 vs. 41.8-74.7%; P < 0.05), but plasma-free leptin concentrations remained unaltered in Cre-Tam mice. We conclude from these results that 1) the relative contribution to whole-body energy metabolism from peripheral leptin signaling is insignificant in vivo, 2) leptin signaling in adipocyte constitutes a distinct short-loop negative feedback regulation of leptin production that is independent of tissue metabolic status, and 3) perturbation of peripheral leptin signaling alone, although increasing leptin production, may not be sufficient to alter the effective plasma levels of leptin because of the counter-regulatory increase in the level of leptin binding protein(s).

Neuronal deletion of Lepr elicits diabesity in mice without affecting cold tolerance or fertility.

Leptin signaling in the brain regulates energy intake and expenditure. To test the degree of functional neuronal leptin signaling required for the maintenance of body composition, fertility, and cold tolerance, transgenic mice expressing Cre in neurons (CaMKIIalpha-Cre) were crossed to mice carrying a floxed leptin receptor (Lepr) allele to generate mice with neuron-specific deletion of Lepr in approximately 50% (C F/F mice) and approximately 75% (C Delta17/F mice) of hypothalamic neurons. Leptin receptor (LEPR)-deficient mice (Delta17/Delta17) with heat-shock-Cre-mediated global Lepr deletion served as obese controls. At 16 wk, male C F/F, C Delta17/F, and Delta17/Delta17 mice were 13.2 (P < 0.05), 45.0, and 55.9% (P < 0.001) heavier, respectively, than lean controls, whereas females showed 31.6, 68.8, and 160.7% increases in body mass (P < 0.001). Significant increases in total fat mass (C F/F: P < 0.01; C Delta17/F and Delta17/Delta17:P < 0.001 vs. sex-matched, lean controls), and serum leptin concentrations (P < 0.001 vs. controls) were present in proportion to Lepr deletion. Male C Delta17/F mice had significant elevations in basal serum insulin concentrations (P < 0.001 vs. controls) and were glucose intolerant, as measured by glucose tolerance test (AUC P < 0.01 vs. controls). In contrast with previous observations in mice null for LEPR signaling, C F/F and C Delta17/F mice were fertile and cold tolerant. These findings support the hypothesis that body weight, adiposity, serum leptin concentrations, and glucose intolerance are proportional to hypothalamic LEPR deficiency. However, fertility and cold tolerance remain intact unless hypothalamic LEPR deficiency is complete.

An allelic series for the leptin receptor gene generated by CRE and FLP recombinase.

Body weight regulation is mediated through several major signaling pathways, some of which have been delineated by positional cloning of spontaneous genetic mutations in mice. Lepr(db/db) mice are obese due to a defect in the signaling portion of the leptin receptor, which has led to extensive study of this highly conserved system over the past several years. We have created an allelic series at Lepr for the further examination of LEPR signaling phenotypes using both the FLP /frt and CRE /loxP systems. By inserting a frt-PGK-neo-frt sequence in Lepr intron 16, we have generated a conditional gene repair Lepr allele ( Lepr-neo) that elicits morbid obesity, diabetes, and infertility in homozygous mice, recapitulating the obesity syndrome of Lepr(db/db) mice. Thus, in vivo excision of the PGK-neo cassette with a FLP recombinase transgene restores the lean and fertile phenotype to Lepr(flox/flox) mice. In the same construct, we have also inserted loxP sites that flank Lepr coding exon 17, a region that encodes a JAK docking site required for STAT3 signaling. CRE-mediated excision of Lepr coding exon 17 from Lepr with a frameshift in subsequent exons results in a syndrome of obesity, diabetes, and infertility in LeprDelta17/Delta17 mice, which is indistinguishable from Lepr(neo/neo) and Lepr(db/db) mice. We conclude that suppression of Lepr gene expression by PGK-neo is phenotypically equivalent to deletion of the Lepr signaling motifs, and therefore the Lepr(neo/neo) mouse may be used to investigate conditional gene repair of Lepr signaling deficiency.

Neuropeptide Y is required for hyperphagic feeding in response to neuroglucopenia.

To investigate the role played by the orexigenic peptide, neuropeptide Y (NPY), in adaptive responses to insulin-induced hypoglycemia, we measured hypothalamic, feeding, and hormonal responses to this stimulus in both wild-type (Npy+/+) and NPY-deficient (Npy-/-) mice. After administration of insulin at a dose (60 mU ip) sufficient to cause moderate hypoglycemia (plasma glucose levels, 40 +/- 3 and 37 +/- 2 mg/dl for Npy+/+ and Npy-/- mice, respectively; P = not significant), 4-h food intake was increased 2.5-fold in Npy+/+ mice relative to saline-injected controls. By comparison, the increase of intake in Npy-/- mice was far smaller (45%) and did not achieve statistical significance (P = 0.08). Hyperphagic feeding in response to insulin-induced hypoglycemia was therefore markedly attenuated in mice lacking NPY, and a similar feeding deficit was detected in these animals after neuroglucopenia induced by 2-deoxyglucose (500 mg/kg ip). A role for NPY in glucoprivic feeding is further supported by our finding that Npy mRNA content (measured by real-time PCR) increased 2.4-fold in the hypothalamus of Npy+/+ mice by 7 h after insulin injection. Unlike the feeding deficits observed in mice lacking NPY, the effect of hypoglycemia to increase plasma glucagon and corticosterone levels was fully intact in these animals, as were both the nadir glucose value and time to recovery of euglycemia after insulin injection (P = not significant). We conclude that NPY signaling is required for hyperphagic feeding, but not neuroendocrine responses to moderate hypoglycemia.

A new obesity-prone, glucose-intolerant rat strain (F.DIO).

Previous breeding for the diet-induced obese (DIO) trait from outbred Sprague-Dawley rats produced a substrain with selection characteristics suggesting a polygenic mode of inheritance. To assess this issue further, selectively bred DIO male rats were crossed with obesity-resistant inbred Fischer F344 dams. Male offspring were crossed twice more against female F344 dams. The resultant N3 (F.DIO) rats were then inbred three more times. On low-fat chow, 10-wk-old male and female DIO rats weighed 86 and 59% more than respective F344 rats. By the N3 (F.DIO) generation, they were only 12 and 10% heavier, respectively. After three additional inbreeding cycles, chow-fed F.DIO males had an exaggerated insulin response to oral glucose compared with F344 rats. After 3 wk on a 31% fat (high-energy) diet, male N3 F.DIO rats gained 16-20% more carcass and adipose weight with 98% higher plasma leptin levels, whereas F.DIO females gained 36-54% more carcass and adipose weight with 130% higher leptin levels than comparable F344 rats. After three inbreeding cycles, F.DIO males still gained more weight on high-energy diet and developed a threefold greater insulin response to oral glucose than F344 males. Preservation of the DIO and glucose intolerance traits through successive backcrosses and inbreeding cycles to produce the F.DIO strain lends further support to the idea that they inherited in a polygenic fashion.