Efficacy of Metreleptin in Obese Patients With Type 2 Diabetes
Efficacy of Metreleptin in Obese Patients With Type 2 Diabetes
Several open-label trials have demonstrated that administering metreleptin to correct overt hypoleptinemia significantly improves the metabolic abnormalities and insulin resistance in patients with congenital lipodystrophy and insulin resistance. Likewise, randomized placebo-controlled trials in patients with human immunodeficiency virus–induced lipodystrophy and the metabolic syndrome who are also hypoleptinemic, although less so than subjects with congenital lipodystrophy, have shown significant, albeit less pronounced, effects of metreleptin. In contrast, obese hyperleptinemic subjects do not respond to exogenously administered leptin. We studied whether metreleptin administration would be effective in hyperleptinemic obese subjects with garden variety insulin resistance and diabetes who are considered leptin-resistant or -tolerant. We observed only minor improvements in the glycemic control of hyperleptinemic/diabetic subjects in this randomized trial, which, albeit statistically significant, are one order of magnitude less pronounced than those observed in hypoleptinemic, lipodystrophic subjects and apparently are not of major clinical importance. Final body weight and levels of inflammatory markers remained completely unaltered in metreleptin-treated hyperleptinemic obese diabetic subjects. This lack of metreleptin's efficacy is consistent with a state of "resistance" or "tolerance" to leptin action, defined as an inability of increasing leptin levels to reduce body weight in obese individuals. This is the first study in obese diabetic subjects that proves the existence of clinical leptin resistance or tolerance similar to the only prior study in obese nondiabetic subjects. Moreover, this study demonstrated no inflammatory response to leptin, with only a minor glycemic response of no apparent clinical significance. Thus, we initiated studies to elucidate mechanisms underlying leptin resistance or tolerance.
We first explored whether mechanisms similar to those involved in other hormone resistance syndromes, such as the increase in binding protein levels, the development of antibodies, the presence of a saturable signaling system, or the presence of in vivo or ex vivo signaling inhibitors in humans, may underlie leptin resistance. A novel finding of this study was that LBP, the extracellular cleaved part of the long isoform of the leptin receptor, and the titers of antileptin antibodies increased significantly in response to metreleptin administration, but not in response to placebo administration. We previously demonstrated that short-term caloric deprivation (72 h) significantly decreases leptin levels but significantly increases LBP by >100%. The current study shows that LBP levels increase in response to several months of pharmacologic metreleptin versus placebo treatment. We also observed for the first time the development of non-neutralizing, leptin–binding antibodies, the titers of which increased in the circulation over time in the majority of subjects treated with metreleptin. The distinct possibility exists that other formulations of leptin that do not have the amino acid (methionine) substitution, which in the case of metreleptin was added to improve protein folding, but have full sequence homology with human leptin may not induce leptin antibodies; this possibility remains to be studied. In addition, because pharmacologic metreleptin doses (~0.2 mg/kg in the current study) may have different effects than physiologic doses in terms of generating antibodies (C.S.M., unpublished data), future placebo–controlled randomized studies involving metreleptin administration in lower, physiologic replacement doses are needed. In any case, the apparent importance of this novel finding is that the majority of circulating leptin is bound to antileptin antibodies, whereas only a small fraction of circulating leptin is free leptin. Thus, we proceeded to explore whether this observation could also be of clinical significance.
Despite increasing LBP and antibody levels, the levels of free leptin increased and remained relatively higher (i.e., at levels ~40–50 ng/mL) in metreleptin-treated subjects. Although these levels are higher than the putative threshold for saturating the blood-brain–barrier leptin transport system, circulating free leptin levels did not correlate with weight loss, indicating clinical ineffectiveness of free leptin levels in the 40–50 ng/mL range. Thus, we decided to study whether metreleptin administration in doses within or above the physiologic leptin range, and encompassing the ~50 ng/mL levels seen in our clinical studies described, can differentially activate signaling pathways in humans in vivo, ex vivo, and in vitro. It has been shown that leptin induces STAT3 phosphorylation in various mouse cell lines and tissues and is generally accepted that this represents a major signaling pathway through which leptin exerts its actions. Despite minor differences in the timing of signaling pathway activation, we observed no major differences in the magnitude of STAT3 activation in response to metreleptin administration in the human peripheral tissues studied in vivo, ex vivo, or in vitro. We observed no differences in leptin-activated signaling pathways when comparing obese versus lean subjects or men versus women in vivo, ex vivo, or in vitro. More important, metreleptin signaling pathways were saturable at a level of ~50 ng/mL, suggesting that above that level, i.e., the level clinically seen in obese subjects at baseline, no additional signaling effect can be observed. This explains the effectiveness of metreleptin in subjects with very low circulating leptin levels and the tolerance to metreleptin's actions when the baseline circulating level is closer to the 40–50 ng/mL range.
We then focused on ER stress, which has recently been shown to play a role in the development of leptin resistance in the hypothalamus of rodents. It has been suggested that ER capacity is directly related to leptin sensitivity, and thus, it has been proposed that ER stress reversal could be used as a strategy to sensitize obese mice and, by extension, humans to leptin. These previous studies have shown that the reduction in ER function creates ER stress, blocks leptin action, and generates leptin resistance in mice, suggesting that ER stress provides a potential mechanism for the development of leptin resistance in which increased ER stress antagonizes and inhibits leptin-mediated STAT3 signaling at a step upstream of STAT3 phosphorylation. Because ER stress cannot yet be studied in humans in vivo, we performed in vitro metreleptin signaling studies in hPAs to explore whether ER stress could underlie the development of leptin resistance in human primary cells. We report for the first time that ER stress limits leptin signaling in hPAs in vitro, indicating that ER stress may induce leptin resistance in humans similar to induction of leptin resistance in mice in vivo and suggesting that improving ER stress could be used as a strategy to sensitize not only obese mice but also humans to metreleptin. Because in vivo leptin actions may differ in comparison with in vitro, studies of in vivo leptin signaling in humans are needed to prove or disprove this hypothesis, but it is currently impossible to perform human in vivo ER stress studies.
The blinded and simultaneous administration of metreleptin or placebo in the current study provides a clear and contemporaneous assessment of the role of metreleptin in regulating body weight, metabolic, and immune function in obese diabetic individuals. It could be argued that metreleptin may have different actions in the pharmacologic range (as used in the current study) than in the physiologic range, in terms of generating antileptin antibodies or possibly by resulting in differential downregulation of leptin receptors. It is unlikely that the latter is the case in the current study because we observed an increase in LBP, which reflects an increased number of cell surface receptors. The possible differences between the effects of pharmacologic and physiologic doses of metreleptin, however, need to be studied by future well-designed clinical trials in which significant weight loss will be induced and replacement dose of metreleptin will be administered and studied in relation to weight maintenance. Finally, although it is difficult to perform in vivo time course signaling experiments in humans, this is an area that also needs to be addressed in future studies. Future in vivo leptin signaling studies involving additional signaling pathways and other peripheral human tissues are also needed. However, leptin signaling in central nervous system tissues (i.e., hypothalami of humans) cannot be studied in vivo or ex vivo, and thus we and others have initiated indirect studies of leptin's actions in the brain using functional magnetic resonance imaging techniques. It is also possible that inducers of leptin resistance other than those in the current study may exist in humans and corresponding leptin sensitizers, if any, remain to be identified.
We believe that beginning to elucidate the mechanisms underlying the physiologic and pharmacologic actions of metreleptin, using studies with direct relevance to humans as those presented in this article, offers distinct advantages over the studies in rodents that have been published to date. These initial translational studies in humans are of direct potential clinical and therapeutic significance.
In summary, in obese patients with diabetes, metreleptin administration for 16 weeks did not alter body weight or circulating inflammatory markers but reduced HbA1c marginally. Furthermore, total leptin, LBP, and antileptin antibody levels increased, limiting free leptin availability and resulting in circulating free leptin levels of ~50 ng/mL. These data identify several steps mediating leptin "tolerance" in humans. Most important, we demonstrate for the first time the saturable nature of leptin signaling pathways in humans and the inhibition of leptin signaling by environmental factors inducing ER stress that contribute significantly to the development of leptin tolerance in obese humans. The mechanisms reported lend themselves to future studies with an ultimate goal of identifying and overcoming leptin resistance in the path toward developing novel therapies for the treatment of excess adiposity and associated abnormalities in humans.
Discussion
Several open-label trials have demonstrated that administering metreleptin to correct overt hypoleptinemia significantly improves the metabolic abnormalities and insulin resistance in patients with congenital lipodystrophy and insulin resistance. Likewise, randomized placebo-controlled trials in patients with human immunodeficiency virus–induced lipodystrophy and the metabolic syndrome who are also hypoleptinemic, although less so than subjects with congenital lipodystrophy, have shown significant, albeit less pronounced, effects of metreleptin. In contrast, obese hyperleptinemic subjects do not respond to exogenously administered leptin. We studied whether metreleptin administration would be effective in hyperleptinemic obese subjects with garden variety insulin resistance and diabetes who are considered leptin-resistant or -tolerant. We observed only minor improvements in the glycemic control of hyperleptinemic/diabetic subjects in this randomized trial, which, albeit statistically significant, are one order of magnitude less pronounced than those observed in hypoleptinemic, lipodystrophic subjects and apparently are not of major clinical importance. Final body weight and levels of inflammatory markers remained completely unaltered in metreleptin-treated hyperleptinemic obese diabetic subjects. This lack of metreleptin's efficacy is consistent with a state of "resistance" or "tolerance" to leptin action, defined as an inability of increasing leptin levels to reduce body weight in obese individuals. This is the first study in obese diabetic subjects that proves the existence of clinical leptin resistance or tolerance similar to the only prior study in obese nondiabetic subjects. Moreover, this study demonstrated no inflammatory response to leptin, with only a minor glycemic response of no apparent clinical significance. Thus, we initiated studies to elucidate mechanisms underlying leptin resistance or tolerance.
We first explored whether mechanisms similar to those involved in other hormone resistance syndromes, such as the increase in binding protein levels, the development of antibodies, the presence of a saturable signaling system, or the presence of in vivo or ex vivo signaling inhibitors in humans, may underlie leptin resistance. A novel finding of this study was that LBP, the extracellular cleaved part of the long isoform of the leptin receptor, and the titers of antileptin antibodies increased significantly in response to metreleptin administration, but not in response to placebo administration. We previously demonstrated that short-term caloric deprivation (72 h) significantly decreases leptin levels but significantly increases LBP by >100%. The current study shows that LBP levels increase in response to several months of pharmacologic metreleptin versus placebo treatment. We also observed for the first time the development of non-neutralizing, leptin–binding antibodies, the titers of which increased in the circulation over time in the majority of subjects treated with metreleptin. The distinct possibility exists that other formulations of leptin that do not have the amino acid (methionine) substitution, which in the case of metreleptin was added to improve protein folding, but have full sequence homology with human leptin may not induce leptin antibodies; this possibility remains to be studied. In addition, because pharmacologic metreleptin doses (~0.2 mg/kg in the current study) may have different effects than physiologic doses in terms of generating antibodies (C.S.M., unpublished data), future placebo–controlled randomized studies involving metreleptin administration in lower, physiologic replacement doses are needed. In any case, the apparent importance of this novel finding is that the majority of circulating leptin is bound to antileptin antibodies, whereas only a small fraction of circulating leptin is free leptin. Thus, we proceeded to explore whether this observation could also be of clinical significance.
Despite increasing LBP and antibody levels, the levels of free leptin increased and remained relatively higher (i.e., at levels ~40–50 ng/mL) in metreleptin-treated subjects. Although these levels are higher than the putative threshold for saturating the blood-brain–barrier leptin transport system, circulating free leptin levels did not correlate with weight loss, indicating clinical ineffectiveness of free leptin levels in the 40–50 ng/mL range. Thus, we decided to study whether metreleptin administration in doses within or above the physiologic leptin range, and encompassing the ~50 ng/mL levels seen in our clinical studies described, can differentially activate signaling pathways in humans in vivo, ex vivo, and in vitro. It has been shown that leptin induces STAT3 phosphorylation in various mouse cell lines and tissues and is generally accepted that this represents a major signaling pathway through which leptin exerts its actions. Despite minor differences in the timing of signaling pathway activation, we observed no major differences in the magnitude of STAT3 activation in response to metreleptin administration in the human peripheral tissues studied in vivo, ex vivo, or in vitro. We observed no differences in leptin-activated signaling pathways when comparing obese versus lean subjects or men versus women in vivo, ex vivo, or in vitro. More important, metreleptin signaling pathways were saturable at a level of ~50 ng/mL, suggesting that above that level, i.e., the level clinically seen in obese subjects at baseline, no additional signaling effect can be observed. This explains the effectiveness of metreleptin in subjects with very low circulating leptin levels and the tolerance to metreleptin's actions when the baseline circulating level is closer to the 40–50 ng/mL range.
We then focused on ER stress, which has recently been shown to play a role in the development of leptin resistance in the hypothalamus of rodents. It has been suggested that ER capacity is directly related to leptin sensitivity, and thus, it has been proposed that ER stress reversal could be used as a strategy to sensitize obese mice and, by extension, humans to leptin. These previous studies have shown that the reduction in ER function creates ER stress, blocks leptin action, and generates leptin resistance in mice, suggesting that ER stress provides a potential mechanism for the development of leptin resistance in which increased ER stress antagonizes and inhibits leptin-mediated STAT3 signaling at a step upstream of STAT3 phosphorylation. Because ER stress cannot yet be studied in humans in vivo, we performed in vitro metreleptin signaling studies in hPAs to explore whether ER stress could underlie the development of leptin resistance in human primary cells. We report for the first time that ER stress limits leptin signaling in hPAs in vitro, indicating that ER stress may induce leptin resistance in humans similar to induction of leptin resistance in mice in vivo and suggesting that improving ER stress could be used as a strategy to sensitize not only obese mice but also humans to metreleptin. Because in vivo leptin actions may differ in comparison with in vitro, studies of in vivo leptin signaling in humans are needed to prove or disprove this hypothesis, but it is currently impossible to perform human in vivo ER stress studies.
The blinded and simultaneous administration of metreleptin or placebo in the current study provides a clear and contemporaneous assessment of the role of metreleptin in regulating body weight, metabolic, and immune function in obese diabetic individuals. It could be argued that metreleptin may have different actions in the pharmacologic range (as used in the current study) than in the physiologic range, in terms of generating antileptin antibodies or possibly by resulting in differential downregulation of leptin receptors. It is unlikely that the latter is the case in the current study because we observed an increase in LBP, which reflects an increased number of cell surface receptors. The possible differences between the effects of pharmacologic and physiologic doses of metreleptin, however, need to be studied by future well-designed clinical trials in which significant weight loss will be induced and replacement dose of metreleptin will be administered and studied in relation to weight maintenance. Finally, although it is difficult to perform in vivo time course signaling experiments in humans, this is an area that also needs to be addressed in future studies. Future in vivo leptin signaling studies involving additional signaling pathways and other peripheral human tissues are also needed. However, leptin signaling in central nervous system tissues (i.e., hypothalami of humans) cannot be studied in vivo or ex vivo, and thus we and others have initiated indirect studies of leptin's actions in the brain using functional magnetic resonance imaging techniques. It is also possible that inducers of leptin resistance other than those in the current study may exist in humans and corresponding leptin sensitizers, if any, remain to be identified.
We believe that beginning to elucidate the mechanisms underlying the physiologic and pharmacologic actions of metreleptin, using studies with direct relevance to humans as those presented in this article, offers distinct advantages over the studies in rodents that have been published to date. These initial translational studies in humans are of direct potential clinical and therapeutic significance.
In summary, in obese patients with diabetes, metreleptin administration for 16 weeks did not alter body weight or circulating inflammatory markers but reduced HbA1c marginally. Furthermore, total leptin, LBP, and antileptin antibody levels increased, limiting free leptin availability and resulting in circulating free leptin levels of ~50 ng/mL. These data identify several steps mediating leptin "tolerance" in humans. Most important, we demonstrate for the first time the saturable nature of leptin signaling pathways in humans and the inhibition of leptin signaling by environmental factors inducing ER stress that contribute significantly to the development of leptin tolerance in obese humans. The mechanisms reported lend themselves to future studies with an ultimate goal of identifying and overcoming leptin resistance in the path toward developing novel therapies for the treatment of excess adiposity and associated abnormalities in humans.
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