Identification and saturable nature of signaling pathways induced by metreleptin in humans: comparative evaluation of in vivo, ex vivo, and in vitro administration

Abstract

Signaling pathways activated by leptin in metabolically important organs have largely been studied only in animal and/or cell culture studies. In this study, we examined whether leptin has similar effects in human peripheral tissues in vivo, ex vivo, and in vitro and whether the response would be different in lean and obese humans. For in vivo leptin signaling, metreleptin was administered and muscle, adipose tissue, and peripheral blood mononuclear cells were taken for analysis of signal activation. Experiments were also done ex vivo and with primary cultured cells in vitro. The signal activation was compared between male versus female and obese versus lean humans. Acute in vivo, ex vivo, and/or in vitro metreleptin administration similarly activated STAT3, AMPK, ERK1/2, Akt, mTOR, NF-κB, and/or IKKα/β without any differences between male versus female and obese versus lean subjects. All signaling pathways were saturable at ∼30-50 ng/mL, consistent with the clinical evidence showing no additional effect(s) in obese subjects who already have high levels of leptin. Our data provide novel information on downstream effectors of metreleptin action in humans that may have therapeutic implications.

Figure 1

Regulation of STAT3, ERK1/2, Akt, and AMPK signaling by in vivo metreleptin administration in hPTs. A–D: Blood draw and fat (lower abdomen) and muscle (thigh) biopsy were performed at baseline. Metreleptin, dose 0.01 mg/kg body weight or placebo (10 cc of normal saline), was given by slow intravenous injection over 1 min, and after 30 min, blood draw and fat and muscle biopsy were performed. Total and phosphorylated STAT3, ERK1/2, Akt, and AMPK were examined by Western blotting. All data were analyzed using Student t test. Values are means (n = 3) ± SD.

Figure 2

Regulation of mTOR signaling by in vivo, ex vivo, and in vitro metreleptin administration in hPTs. A: In vivo signaling in subcutaneous fat, muscle, and PBMCs before and 30 min after metreleptin administration in female vs. male and lean vs. obese subjects. B: Signaling in subcutaneous (SC) and omental (OM) fat from female obese subjects before and 30 min after ex vivo metreleptin administration. C: Time- and dose-dependent signaling by in vitro metreleptin administration in primary human adipocytes from female obese subjects. D: Time- and dose-dependent signaling in hPBMCs from three female obese subjects by ex vivo metreleptin administration. E: Signaling in muscle from three female obese subjects before and 30 min after ex vivo metreleptin administration. F: Time- and dose-dependent signaling in primary muscle cells from three female obese subjects by in vitro metreleptin administration. Total and phosphorylated mTOR were examined by Western blotting. All data were analyzed using Student t test and/or 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, whereas means with similar letters are not different from each other. C, control.

Figure 3

Regulation of NF-κB signaling by ex vivo and in vitro metreleptin administration in hPTs. A: Signaling in subcutaneous (SC) and omental (OM) fat from three female obese subjects before and 30 min after ex vivo metreleptin administration. B: Time- and dose-dependent signaling by in vitro metreleptin administration in primary human adipocytes from three female obese subjects. C: Time- and dose-dependent signaling in hPBMCs from three female obese subjects by ex vivo metreleptin administration. D: Signaling in muscle from three female obese subjects before and 30 min after ex vivo metreleptin administration. E: Time- and dose-dependent signaling in primary muscle cells from three female obese subjects by in vitro metreleptin administration. Total and phosphorylated NF-κB were examined by Western blotting. All data were analyzed using Student t test and/or 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, whereas means with similar letters are not different from each other. C, control.

Figure 4

Regulation of IKKα/β signaling by ex vivo and in vitro metreleptin administration in hPTs. A: Signaling in subcutaneous (SC) and omental (OM) fat from three female obese subjects before and 30 min after ex vivo metreleptin administration. B: Time- and dose-dependent signaling by in vitro metreleptin administration in primary human adipocytes from three female obese subjects. C: Time- and dose-dependent signaling in hPBMCs from three female obese subjects by ex vivo metreleptin administration. D: Signaling in muscle from three female obese subjects before and 30 min after ex vivo metreleptin administration. E: Time- and dose-dependent signaling in primary muscle cells from three female obese subjects by in vitro metreleptin administration. Total and phosphorylated IKKα/β were examined by Western blotting. All data were analyzed using Student t test and/or 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, whereas means with similar letters are not different from each other. C, control.

Figure 5

Regulation of SHP2 signaling by in vivo and ex vivo metreleptin administration in hPTs. A: Signaling in female/male, lean/obese subjects before and 30 min after in vivo metreleptin administration. B: Signaling in subcutaneous (SC) and omental (OM) fat from three female obese subjects before and 30 min after ex vivo metreleptin administration. C: Time- and dose-dependent signaling in hPBMCs from three female obese subjects by ex vivo metreleptin administration. Total and phosphorylated SHP2 were examined by Western blotting. All data were analyzed using Student t test and/or 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, whereas means with similar letters are not different from each other. C, control.

Figure 6

Regulation of differentiation and lipolysis by in vitro metreleptin administration in hPAs. A: Primary cultured subcutaneous (SC) and omental (OM) adipocytes were treated with metreleptin (50 ng/mL) in vitro for 28 days, and Oil Red O staining was then performed. B: Cell differentiation rate was quantified by extraction of Oil Red O with isopropanol. C: SC and OM adipocytes were treated with metreleptin (50 ng/mL) in vitro for 36 h. Lipolysis was measured using [¹⁴C]oleic acid method. Isoproterenol (ISO, 10 μmol/L for 30 min) was used as a positive control. All 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, whereas means with similar letters are not different from each other. C, control; D, day; L, leptin.

Figure 7

Regulation of cell growth signaling and hypertrophy by in vitro metreleptin administration in CMs and USMs. A: The cells were treated with metreleptin (50 ng/mL) in vitro for 30 min with/without AG490 (STAT3 inhibitor, 1 μmol/L for 1 h). B: The cells were treated with metreleptin (50 ng/mL) in vitro for 30 min. Total and phosphorylated STAT3, ERK, JNK, and p38 were examined by Western blotting. C: The cells were treated with metreleptin (50 ng/mL) in vitro for 36 h, and hypertrophy assay was then performed using 0.1% rhodamine-phalloidin. All data were analyzed using Student t test and/or 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, whereas means with the same letters are not different from each other. C, control; L, leptin.