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Introduction to Leptin

  • Definition: Leptin is a protein product of the ob gene.

  • Source: Predominantly secreted from white adipose tissue.

  • Function: Regulates:

    • Food intake

    • Energy expenditure

    • Neuroendocrine function (References 1–4).

Leptin in Genetically Obese Mice

  • In ob/ob mice (genetically obese due to lack of functional leptin), the administration of recombinant leptin is effective in reversing obesity (References 2, 3, 5).

  • Leptin resistance exists in most cases of obesity in both rodents and humans with high circulating leptin levels (References 6, 7).

Mechanisms of Leptin Resistance

  • Potential sites for leptin resistance include:

    • Blood-brain barrier transport system (References 8, 9).

    • Leptin signaling mechanism in leptin-responsive neurons in the hypothalamus (Reference 10).

  • In diet-induced obesity (DIO) in rodents, an increased plasma leptin level leads to resistance against the effects of exogenous leptin administered peripherally (References 11, 12).

  • However, these rodents respond to central (icv) administration of leptin, indicating a defect in the blood-brain barrier.

Hypothalamic STAT3 Signaling

  • When leptin is administered peripherally to normal mice, it activates hypothalamic signaling through the STAT3 pathway.

  • Methods:

    • Animals and Diets: Male C57BL/6J mice were bred and housed in standard conditions.

    • At 4 weeks, mice were divided into two diet groups:

    • High-fat diet (45% fat).

    • Low-fat diet (10% fat).

    • Body weight and food intake were measured weekly.

Experimental Procedures

  • After specific timepoints, mice received central and peripheral injections of leptin or buffer.

  • Mice were then anesthetized and sacrificed to dissect hypothalami for assays (EMSA and RT-PCR).

Hormonal Assays

  • Recombinant mouse leptin preparation:

    • Dissolved in PBS (pH 7.4) with 0.1% BSA, for ip or icv injections.

  • Plasma leptin levels were evaluated using radioimmunoassay (Linco Research Inc.).

Quantitative RT-PCR

  • Primers used for specific PCR amplification:

    • SOCS-3: 5′-ACCAGCGCCACTTCTTCACG-3′ / 5′-GTGGAGCATCATACTGATCC-3′

    • PIAS-3: 5′-AGGAGAAACTGACTGCTGACC-3′ / 5′-AATCAGCGACTCATAGGGAGCC-3′

    • STAT3: 5′-TCCTCTATCAGCACAACCTTCG-3′ / 5′-AGCTGCTGCATCTTCTGTCTGG-3′

    • ObRa: 5′-ACACTGTTAATTTCAACACCAGAG-3′ / 5′-AGTCATTCAAACCATTAGTTTAGG-3′

    • ObRb: 5′-GGGTAATACTTAAACAGTGACC-3′ / 5′-CTATCTGAAAATAAAAACTTCATG-3′

    • β-actin: 5′-CGTACCACGGGCATTGTGATGG-3′ / 5′-TTTGATGTACACGCACGATTTCCC-3′

Results and Discussion

  • The objective of the study was to elucidate the mechanisms of leptin resistance during high-fat diet in C57BL/6J mice.

  • Leptin Administration: Mice received leptin via ip or icv routes, and STAT3 activation was assessed in the hypothalamus.

  • Dose-Response:

    • A dose-response study highlighted that icv administration was more effective than ip, with half-maximal doses indicating a 30-fold increased potency when given icv.

    • Dose for icv: 0.5 µg; Dose for ip: 15 µg.

  • Physiological Changes:

    • Mice on the high-fat diet gained weight significantly after 4 days and weighed 24% more than low-fat counterparts after 15 weeks.

    • Increased caloric intake observed for high-fat diet mice.

Leptin Signaling

  • 4-Week Insights: In the initial 4 weeks, both diet groups exhibited similar peripheral leptin signaling.

  • 15-Week Insights: Mice on the high-fat diet showed significant leptin resistance, with no observable STAT3 activity from ip leptin.

  • Although peripheral leptin failed to induce signaling, icv administration retained some level of effectiveness but at a much lower magnitude compared to low-fat fed mice.

Mechanisms of Resistance

  • Two defects identified in leptin resistance:

    1. Transport Defect: Ineffective peripheral access to hypothalamus due to blood-brain barrier dysfunction.

    2. Signaling Defect: Impaired intracellular signaling affecting leptin-responsive neurons, specifically STAT3 activation.

Additional Research Areas

  • Future research aims to uncover the precise molecular mechanisms of decreased leptin actions and the implications of these findings toward treatments for obesity, particularly concerning how leptin mimics could be developed for effective human obesity treatment.

Acknowledgments

  • This work supported by grants from NIH, Eli Lilly, Swiss National Scientific Research Foundation, Joslin Diabetes Center, and Deutsche Forschungsgemeinschaft.