Efficacy of Metreleptin in Obese Patients With Type 2 Diabetes
Circulating leptin levels increased significantly in men and women treated with metreleptin over the 4-month study period (Table 1, Supplementary Appendix 1). Non-neutralizing antileptin antibodies developed with increasing titers, and LBP levels decreased less in metreleptin-treated subjects compared with placebo-treated subjects. BMI remained unchanged throughout the study in both treatment groups. Free leptin levels were similar in both treatment groups at baseline but increased significantly in subjects treated with metreleptin despite the presence of antibodies against metreleptin. Although the development of antileptin antibodies was significantly and positively correlated with the increase in total circulating leptin levels (r = 0.99, P < 0.0001), circulating free leptin levels (r = 0.44, P = 0.012, SI 2) were also increased. This indicates that increasing doses of leptin can break through the resistance caused by increasing binding of leptin by leptin antibodies. The increase in free leptin levels to ~48.4 ng/mL was not associated with any significant differential weight loss or changes in inflammatory markers in metreleptin-treated patients and resulted in only a small but differential improvement in glycemic control (HbA1c from 8.01 ± 0.93 to 7.96 ± 1.12, P = 0.03). Changes of leptin and free leptin levels were not correlated with changes of body weight or inflammatory markers studied (Table 2). Baseline TNFR-II and monocyte chemoattractant protein-1 levels were relatively similar at baseline in both treatment groups but increased in men and women treated with metreleptin. By contrast, IL-6, IL-10, CRP, sTNFR-I, and soluble intracellular adhesive molecule-I did not change significantly in any group (Table 1).
We asked whether the antibodies produced after treatment with metreleptin in clinical study I were able to affect (stimulate or neutralize) leptin-related proliferation and intracellular signaling. Thus, we used the BAF3 cell lines after transfection with the long form of the human leptin receptor (hLepRBAF3). First, we developed standard curves of antileptin antibodies using an antileptin mAb generated in the laboratory (Supplementary Appendix 2A). Antibodies isolated from metreleptin-treated obese subjects were able to stimulate proliferation up to ~50-fold more than leptin alone but only at lower leptin concentrations (Fig. 1A). On the contrary, no additional stimulatory effect was observed in the same subjects before treatment, in the placebo-treated subjects, or when using IgGs from normal subjects (Fig. 1B and C). These functional effects were also analyzed at the signaling level in terms of STAT3 phosphorylation. IgGs from metreleptin-treated obese subjects significantly increased p-STAT3 levels (Fig. 1D, Supplementary Appendix 2B), whereas no effect was observed after addition of either placebo-treated IgGs or IgGs isolated from normal controls (Fig. 1E and F, Supplementary Appendix 2B). These results suggest that metreleptin treatment results in the production of antileptin antibodies that are not neutralizing but may demonstrate minor agonistic activities on hLepR measured both as increased proliferation of a leptin-dependent cell line and as increased hLepR-mediated STAT3 signaling. The stimulatory effect of these antibodies is small, however, and easily overridden by relatively higher leptin doses.
(Enlarge Image)
Figure 1.
Laboratory study I. Agonistic/stimulatory activity of antileptin antibodies generated during metreleptin administration. A–C: The functional activity of antileptin antibodies in hLepRBAF3 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 hLepRBAF3 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.
To study whether intracellular signaling pathways are differentially activated (and thus underlie leptin resistance) in leptin-sensitive lean subjects versus leptin-resistant obese subjects in response to increasing leptin levels similar to those seen in clinical trials, we performed in vivo signaling experiments. In vivo metreleptin administration (0.01 mg/kg) induced an approximately 3.2-fold increase in p-STAT3 at 30 min versus baseline in both hAT (Supplementary Appendix 3A) and hPBMCs (Supplementary Appendix 3B), with no significant difference in p-STAT3 levels from lean versus obese subjects.
No Differential Activation of STAT3 Signaling by Ex Vivo Metreleptin Administration in hAT and hPBMC. Dose-response curves showed that administration of up to 50 ng/mL metreleptin for 30 min significantly induced phosphorylation of STAT3 in subcutaneous and omental hAT from obese male and female subjects (Fig. 2A and B). These results were similar to what was observed in hAT and hPBMCs after in vivo metreleptin administration (Supplementary Appendix 3AandB). Similar to in vivo observations, there was no significant difference in p-STAT3 expression in hAT from obese versus lean subjects (Fig. 2C and D). The phosphorylated form of STAT3 was increased in metreleptin-stimulated hPBMCs (Fig. 2E and F) compared with control, showing that activation was evident as early as 5 min after ex vivo stimulation with metreleptin.
(Enlarge Image)
Figure 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.
No Differential Activation of MAPK Signaling by Ex Vivo Metreleptin Administration in hAT. Ex vivo metreleptin administration stimulated activation of MAPK by ~3.1-fold in hAT (Fig. 2G) from obese male and female subjects. There was no difference in MAPK activation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
No Differential Expression of Inhibitors of Ex Vivo Metreleptin Signaling in hAT. We observed no activation of SOCS3 by ex vivo metreleptin administration in hAT (Supplementary Appendix 4). There was no difference in SOCS3 activation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
No Differential Activation of AMPK Signaling by Ex Vivo Metreleptin Administration in hAT and hPBMC. Ex vivo metreleptin administration stimulated phosphorylation of AMPK in both subcutaneous and omental hAT, and hPBMCs from obese female subjects (Supplementary Appendix 5). We observed no difference in AMPK activation from subcutaneous versus omental, obese versus lean, and male versus female subjects.
No Differential Activation of STAT3 Signaling by In Vitro Metreleptin Administration in hPA. In vitro metreleptin administration significantly induced phosphorylation of STAT3 by ~2.1-fold at 10 min with a trend toward greater induction at 20–40 min (Fig. 3A and B). A considerable amount of p-STAT3 in metreleptin-treated cells, but only background levels of p-STAT3 in control cells, was detected in both subcutaneous and omental hPA (Fig. 3C). However, these effects were totally blocked by pretreatment with AG490, a STAT3 inhibitor (Fig. 3D), suggesting that metreleptin stimulation activates STAT3 signaling in hPA. In addition, we observed nuclear translocation of STAT3 by in vitro metreleptin administration in a dose-dependent manner (Supplementary Appendix 6). Metreleptin signaling pathways in hPA were saturable at a level of ~50 ng/mL, and these results are consistent with those observed in hAT in vivo and ex vivo. There were no significant differences in STAT3 activation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
(Enlarge Image)
Figure 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.)
No Differential Activation of AMPK Signaling by In Vitro Metreleptin Administration in hPA. In vitro metreleptin administration increased AMPK phosphorylation by ~2.5-fold in both subcutaneous and omental hPA from male and female subjects (Fig. 3E). We observed no differences in AMPK phosphorylation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
No Differential Expression of Inhibitors of Metreleptin Signaling in hPA. We observed no early activation of SOCS3 by in vitro metreleptin administration in hPA (Fig. 3F). In addition, we observed no differences in expression of inhibitors from subcutaneous versus omental, obese versus lean, and male versus female subjects.
Downregulation of In Vitro Metreleptin-stimulated STAT3 Signaling by ER Stress in hPA. Stimulation of the cells with metreleptin led to a marked increase in phosphorylation of STAT3, but when challenged with ER stress (tunicamycin and dithiothreitol), the metreleptin-activated STAT3 phosphorylation was abolished totally (Supplementary Appendix 7). We observed no differences in ER stress–mediated STAT3 phosphorylation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
To validate our in vivo, ex vivo, and in vitro metreleptin signaling data, we used publicly available datasets to examine expression of potential inhibitors of leptin signaling in hAT from lean and obese nondiabetic humans. We analyzed a dataset comparing gene expression profiles from subcutaneous adipocytes from 20 lean and 19 obese nondiabetic Pima Indians, and there was no significant difference in the expression levels of PTP1B or SOCS3.
Results
Clinical study I: Body Weight, Metabolic, and Immune Responses to Metreleptin Versus Placebo Treatment in Obese Hyperleptinemic Subjects with Diabetes
Circulating leptin levels increased significantly in men and women treated with metreleptin over the 4-month study period (Table 1, Supplementary Appendix 1). Non-neutralizing antileptin antibodies developed with increasing titers, and LBP levels decreased less in metreleptin-treated subjects compared with placebo-treated subjects. BMI remained unchanged throughout the study in both treatment groups. Free leptin levels were similar in both treatment groups at baseline but increased significantly in subjects treated with metreleptin despite the presence of antibodies against metreleptin. Although the development of antileptin antibodies was significantly and positively correlated with the increase in total circulating leptin levels (r = 0.99, P < 0.0001), circulating free leptin levels (r = 0.44, P = 0.012, SI 2) were also increased. This indicates that increasing doses of leptin can break through the resistance caused by increasing binding of leptin by leptin antibodies. The increase in free leptin levels to ~48.4 ng/mL was not associated with any significant differential weight loss or changes in inflammatory markers in metreleptin-treated patients and resulted in only a small but differential improvement in glycemic control (HbA1c from 8.01 ± 0.93 to 7.96 ± 1.12, P = 0.03). Changes of leptin and free leptin levels were not correlated with changes of body weight or inflammatory markers studied (Table 2). Baseline TNFR-II and monocyte chemoattractant protein-1 levels were relatively similar at baseline in both treatment groups but increased in men and women treated with metreleptin. By contrast, IL-6, IL-10, CRP, sTNFR-I, and soluble intracellular adhesive molecule-I did not change significantly in any group (Table 1).
Laboratory Study I: Agonistic/Stimulatory Activity of Antileptin Antibodies Generated During Metreleptin Administration
We asked whether the antibodies produced after treatment with metreleptin in clinical study I were able to affect (stimulate or neutralize) leptin-related proliferation and intracellular signaling. Thus, we used the BAF3 cell lines after transfection with the long form of the human leptin receptor (hLepRBAF3). First, we developed standard curves of antileptin antibodies using an antileptin mAb generated in the laboratory (Supplementary Appendix 2A). Antibodies isolated from metreleptin-treated obese subjects were able to stimulate proliferation up to ~50-fold more than leptin alone but only at lower leptin concentrations (Fig. 1A). On the contrary, no additional stimulatory effect was observed in the same subjects before treatment, in the placebo-treated subjects, or when using IgGs from normal subjects (Fig. 1B and C). These functional effects were also analyzed at the signaling level in terms of STAT3 phosphorylation. IgGs from metreleptin-treated obese subjects significantly increased p-STAT3 levels (Fig. 1D, Supplementary Appendix 2B), whereas no effect was observed after addition of either placebo-treated IgGs or IgGs isolated from normal controls (Fig. 1E and F, Supplementary Appendix 2B). These results suggest that metreleptin treatment results in the production of antileptin antibodies that are not neutralizing but may demonstrate minor agonistic activities on hLepR measured both as increased proliferation of a leptin-dependent cell line and as increased hLepR-mediated STAT3 signaling. The stimulatory effect of these antibodies is small, however, and easily overridden by relatively higher leptin doses.
(Enlarge Image)
Figure 1.
Laboratory study I. Agonistic/stimulatory activity of antileptin antibodies generated during metreleptin administration. A–C: The functional activity of antileptin antibodies in hLepRBAF3 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 hLepRBAF3 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.
Clinical Study II: In Vivo Metreleptin Signaling in hAT and hPBMCs from Lean and Obese Subjects
To study whether intracellular signaling pathways are differentially activated (and thus underlie leptin resistance) in leptin-sensitive lean subjects versus leptin-resistant obese subjects in response to increasing leptin levels similar to those seen in clinical trials, we performed in vivo signaling experiments. In vivo metreleptin administration (0.01 mg/kg) induced an approximately 3.2-fold increase in p-STAT3 at 30 min versus baseline in both hAT (Supplementary Appendix 3A) and hPBMCs (Supplementary Appendix 3B), with no significant difference in p-STAT3 levels from lean versus obese subjects.
Laboratory Study II: Comparative Evaluation of Ex Vivo Metreleptin Signaling in hAT and hPBMCs from Lean and Obese Subjects
No Differential Activation of STAT3 Signaling by Ex Vivo Metreleptin Administration in hAT and hPBMC. Dose-response curves showed that administration of up to 50 ng/mL metreleptin for 30 min significantly induced phosphorylation of STAT3 in subcutaneous and omental hAT from obese male and female subjects (Fig. 2A and B). These results were similar to what was observed in hAT and hPBMCs after in vivo metreleptin administration (Supplementary Appendix 3AandB). Similar to in vivo observations, there was no significant difference in p-STAT3 expression in hAT from obese versus lean subjects (Fig. 2C and D). The phosphorylated form of STAT3 was increased in metreleptin-stimulated hPBMCs (Fig. 2E and F) compared with control, showing that activation was evident as early as 5 min after ex vivo stimulation with metreleptin.
(Enlarge Image)
Figure 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.
No Differential Activation of MAPK Signaling by Ex Vivo Metreleptin Administration in hAT. Ex vivo metreleptin administration stimulated activation of MAPK by ~3.1-fold in hAT (Fig. 2G) from obese male and female subjects. There was no difference in MAPK activation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
No Differential Expression of Inhibitors of Ex Vivo Metreleptin Signaling in hAT. We observed no activation of SOCS3 by ex vivo metreleptin administration in hAT (Supplementary Appendix 4). There was no difference in SOCS3 activation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
No Differential Activation of AMPK Signaling by Ex Vivo Metreleptin Administration in hAT and hPBMC. Ex vivo metreleptin administration stimulated phosphorylation of AMPK in both subcutaneous and omental hAT, and hPBMCs from obese female subjects (Supplementary Appendix 5). We observed no difference in AMPK activation from subcutaneous versus omental, obese versus lean, and male versus female subjects.
Laboratory Study III: In Vitro Metreleptin Signaling in Subcutaneous and Omental hPAs from Lean and Obese Subjects
No Differential Activation of STAT3 Signaling by In Vitro Metreleptin Administration in hPA. In vitro metreleptin administration significantly induced phosphorylation of STAT3 by ~2.1-fold at 10 min with a trend toward greater induction at 20–40 min (Fig. 3A and B). A considerable amount of p-STAT3 in metreleptin-treated cells, but only background levels of p-STAT3 in control cells, was detected in both subcutaneous and omental hPA (Fig. 3C). However, these effects were totally blocked by pretreatment with AG490, a STAT3 inhibitor (Fig. 3D), suggesting that metreleptin stimulation activates STAT3 signaling in hPA. In addition, we observed nuclear translocation of STAT3 by in vitro metreleptin administration in a dose-dependent manner (Supplementary Appendix 6). Metreleptin signaling pathways in hPA were saturable at a level of ~50 ng/mL, and these results are consistent with those observed in hAT in vivo and ex vivo. There were no significant differences in STAT3 activation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
(Enlarge Image)
Figure 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.)
No Differential Activation of AMPK Signaling by In Vitro Metreleptin Administration in hPA. In vitro metreleptin administration increased AMPK phosphorylation by ~2.5-fold in both subcutaneous and omental hPA from male and female subjects (Fig. 3E). We observed no differences in AMPK phosphorylation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
No Differential Expression of Inhibitors of Metreleptin Signaling in hPA. We observed no early activation of SOCS3 by in vitro metreleptin administration in hPA (Fig. 3F). In addition, we observed no differences in expression of inhibitors from subcutaneous versus omental, obese versus lean, and male versus female subjects.
Downregulation of In Vitro Metreleptin-stimulated STAT3 Signaling by ER Stress in hPA. Stimulation of the cells with metreleptin led to a marked increase in phosphorylation of STAT3, but when challenged with ER stress (tunicamycin and dithiothreitol), the metreleptin-activated STAT3 phosphorylation was abolished totally (Supplementary Appendix 7). We observed no differences in ER stress–mediated STAT3 phosphorylation from subcutaneous versus omental, male versus female, and obese versus lean subjects.
Laboratory Study IV: Gene Expression Omnibus Datasets Analysis of Published Data on Expression of Inhibitors of Metreleptin Signaling
To validate our in vivo, ex vivo, and in vitro metreleptin signaling data, we used publicly available datasets to examine expression of potential inhibitors of leptin signaling in hAT from lean and obese nondiabetic humans. We analyzed a dataset comparing gene expression profiles from subcutaneous adipocytes from 20 lean and 19 obese nondiabetic Pima Indians, and there was no significant difference in the expression levels of PTP1B or SOCS3.
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