Efficacy of metreleptin in obese patients with type 2 diabetes: cellular and molecular pathways underlying leptin tolerance

Abstract

Objective: Metreleptin has been efficacious in improving metabolic control in patients with lipodystrophy, but its efficacy has not been tested in obese patients with type 2 diabetes.

Research design and methods: We studied the role of leptin in regulating the endocrine adaptation to long-term caloric deprivation and weight loss in obese diabetic subjects over 16 weeks in the context of a double-blinded, placebo-controlled, randomized trial. We then performed detailed interventional and mechanistic signaling studies in humans in vivo, ex vivo, and in vitro.

Results: In obese patients with diabetes, metreleptin administration for 16 weeks did not alter body weight or circulating inflammatory markers but reduced HbA(1c) marginally (8.01 ± 0.93-7.96 ± 1.12, P = 0.03). Total leptin, leptin-binding protein, and antileptin antibody levels increased, limiting free leptin availability and resulting in circulating free leptin levels of ∼50 ng/mL. Consistent with clinical observations, all metreleptin signaling pathways studied in human adipose tissue and peripheral blood mononuclear cells were saturable at ∼50 ng/mL, with no major differences in timing or magnitude of leptin-activated STAT3 phosphorylation in tissues from male versus female or obese versus lean humans in vivo, ex vivo, or in vitro. We also observed for the first time that endoplasmic reticulum (ER) stress in human primary adipocytes inhibits leptin signaling.

Conclusions: In obese patients with diabetes, metreleptin administration did not alter body weight or circulating inflammatory markers but reduced HbA(1c) marginally. ER stress and the saturable nature of leptin signaling pathways play a key role in the development of leptin tolerance in obese patients with diabetes.

FIG. 1.

Laboratory study I. Agonistic/stimulatory activity of antileptin antibodies generated during metreleptin administration. A–C: The functional activity of antileptin antibodies in hLepR⁺BAF3 cells was as described in detail in research design and methods. ■, Leptin + IgG posttreatment; ♦, Leptin + IgG pretreatment. D–F: The biochemical level of the capacity of antileptin IgGs in hLepR⁺BAF3 cells was studied as described in detail in research design and methods. All density values for each protein band of interest are expressed as a fold increase. Data were analyzed using one-way ANOVA followed by post hoc test for multiple comparisons. Values are means (n = 6) ± SD. Means with different letters are significantly different, P < 0.05. L.N.C., lean normal control.

FIG. 2.

Laboratory study II. Comparative evaluation of ex vivo metreleptin signaling in hAT and hPBMCs from lean and obese subjects. Ex vivo metreleptin administration in hAT and hPBMCs was performed as described in detail in research design and methods. hAT (A–D) and hPBMCs (E) were incubated and stimulated with or without ex vivo metreleptin at the indicated concentrations for 30 min. F: hPBMCs were incubated and stimulated with or without ex vivo metreleptin at the indicated times. G: hAT was incubated and stimulated with or without ex vivo metreleptin at the indicated concentrations for 30 min. All lysates were examined by Western blotting as described in detail in research design and methods. All density values for each protein band of interest are expressed as a fold increase. Data were analyzed using one-way ANOVA followed by post hoc test for multiple comparisons. Values are means (n = 3) ± SD. Means with different letters are significantly different, P < 0.05. OM, omental; SC, subcutaneous.

FIG. 3.

Laboratory study III. In vitro metreleptin signaling in subcutaneous (SC) and omental (OM) hPA from lean and obese subjects. In vitro metreleptin administration in hPA was performed as described in detail in research design and methods. A: Cells were treated with metreleptin at the indicated concentrations for 30 min. B: Cells were treated with metreleptin at the indicated times. C: Cells were treated with metreleptin (50 ng/mL) for 30 min. Immunodetection was carried out as described in detail in research design and methods. All pictures were ×40 magnification. D: Cells were pretreated with the STAT3 inhibitor AG490 (AG, 1 μmol/L) for 1 h, followed by treatment with 50 ng/mL metreleptin for 30 min. E and F: Cells were treated with metreleptin at the indicated concentrations for 30 min. All lysates were examined by Western blotting as described in detail in research design and methods. All density values for each protein band of interest are expressed as a fold increase. Data were analyzed using one-way ANOVA followed by post hoc test for multiple comparisons. Values are means (n = 3) ± SD. Means with different letters are significantly different, P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)