Estrogenic activity of E2-conjugated GLP-1 is mediated by intracellular endolysosomal acidification and estrone metabolism

IF 7 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism Pub Date : 2025-04-07 DOI:10.1016/j.molmet.2025.102136

Callum Coupland , Na Sun , Ahmed Khalil , Özüm Ezgi Karaoglu , Arkadiusz Liskiewicz , Daniela Liskiewicz , Gerald Grandl , Seun Akindehin , Gandhari Maity , Bin Yang , Brian Finan , Patrick Knerr , Jonathan D. Douros , Axel Walch , Richard DiMarchi , Matthias H. Tschöp , Timo D. Müller , Aaron Novikoff

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引用次数: 0

Abstract

Objective

Recent modifications to glucagon-like peptide 1 (GLP-1), known for its insulinotropic and satiety-inducing effects, have focused on conjugating small molecules to enable selective delivery into GLP-1R+ tissues to achieve targeted synergy and improved metabolic outcomes. Despite continued advancements in GLP-1/small molecule conjugate strategies, the intracellular mechanisms facilitating concurrent GLP-1R signaling and small molecule cargo release remain poorly understood.

Methods

We evaluate an estradiol (E2)-conjugated GLP-1 (GLP-1-CEX/E2) for relative differences in GLP-1R signaling and trafficking, and elucidate endolysosomal dynamics that lead to estrogenic activity using various live-cell, reporter, imaging, and mass-spectrometry techniques.

Results

We find GLP-1-CEX/E2 does not differentially activate or traffic the GLP-1R relative to its unconjugated GLP-1 backbone (GLP-1-CEX), but uniquely internalizes the E2 moiety and stimulates estrogenic signaling. Endolysosomal pH-dependent proteolytic activity likely mediates E2 moiety liberation, as evidenced by clear amplification in estrogenic activity following co-administration with lysosomal VATPase activator EN6. The hypothesized liberated metabolite from GLP-1-CEX/E2, E2-3-ether, exhibits partial estrogenic efficacy through ERα, and is predisposed toward estrone-3-sulfate conversion. Finally, we identify relative increases in intracellular E2, estrone, and estrone-3-sulfate following GLP-1-CEX/E2 incubation in GLP-1R+ cells, demonstrating proof-of-principle for desired cargo release.

Conclusion

Together, our data suggest that GLP-1-CEX/E2 depends on GLP-1R trafficking and lysosome acidification for estrogenic efficacy, with a likely conversion of the liberated E2-3-ether metabolite into estrone-3-sulfate, resulting in a residual downstream flux into active estradiol. Our current findings aim to improve the understanding of small molecule targeting and the efficacy behind GLP-1/small molecule conjugates.

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e2偶联GLP-1的雌激素活性是由细胞内溶酶体酸化和雌激素代谢介导的

胰高血糖素样肽1 （GLP-1）以其促胰岛素和诱导饱腹感作用而闻名，最近对其的修饰主要集中在偶联小分子上，使其能够选择性地递送到GLP-1R+组织中，以实现靶向协同作用并改善代谢结果。尽管GLP-1/小分子偶联策略不断取得进展，但细胞内促进GLP-1R信号传导和小分子货物释放的机制仍然知之甚少。方法我们评估了雌二醇（E2）偶联GLP-1 （GLP-1- cex /E2）在GLP-1R信号传导和运输方面的相对差异，并利用各种活细胞、报告细胞、成像和质谱技术阐明了导致雌激素活性的内溶酶体动力学。结果我们发现GLP-1- cex /E2与GLP-1主干（GLP-1- cex）相比，对GLP-1R的激活或转运没有差异，而是独特地内化E2部分并刺激雌激素信号传导。内溶酶体ph依赖性蛋白水解活性可能介导E2部分的释放，与溶酶体VATPase激活剂EN6共同给药后雌激素活性明显扩增。假设从GLP-1-CEX/E2中释放的代谢物E2-3-醚通过ERα表现出部分雌激素功效，并倾向于雌激素-3-硫酸盐转化。最后，我们确定了GLP-1-CEX/E2在GLP-1R+细胞中孵卵后细胞内E2、雌酮和雌酮-3-硫酸的相对增加，证明了所需货物释放的原理证明。综上所述，我们的数据表明，GLP-1-CEX/E2依赖于GLP-1R的转运和溶酶体的酸化来实现雌激素的功效，释放的E2-3-醚代谢物可能转化为雌酮-3-硫酸盐，导致剩余的下游通量转化为活性雌二醇。我们目前的研究结果旨在提高对小分子靶向和GLP-1/小分子偶联物功效的理解。

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来源期刊

Molecular Metabolism ENDOCRINOLOGY & METABOLISM-

CiteScore

14.50

自引率

2.50%

发文量

219

审稿时长

43 days

期刊介绍： Molecular Metabolism is a leading journal dedicated to sharing groundbreaking discoveries in the field of energy homeostasis and the underlying factors of metabolic disorders. These disorders include obesity, diabetes, cardiovascular disease, and cancer. Our journal focuses on publishing research driven by hypotheses and conducted to the highest standards, aiming to provide a mechanistic understanding of energy homeostasis-related behavior, physiology, and dysfunction. We promote interdisciplinary science, covering a broad range of approaches from molecules to humans throughout the lifespan. Our goal is to contribute to transformative research in metabolism, which has the potential to revolutionize the field. By enabling progress in the prognosis, prevention, and ultimately the cure of metabolic disorders and their long-term complications, our journal seeks to better the future of health and well-being.